To close or not to close

Abstract

Regulation of symplastic communication through plasmodesmata (PD) is crucial for coordinating physiological and developmental processes in plants. Recently, we have demonstrated that the reduction of plasmodesmal connectivity by plasmodesmata-located protein 5 (PDLP5) is linked to an enhanced defense response against bacterial pathogens in Arabidopsis.¹ One very intriguing aspect of PDLP5 is that highly-increased PDLP5 expression causes positive feedback amplification of SA, which is manifested by spontaneous formation of hypersensitive-response (HR)-like lesions. Although the underlying mechanism has yet to be uncovered, it is conceivable that some sort of signal relay or messenger system may operate between the PD and the nucleus and/or other compartments to bring about the observed changes in hormonal level and cellular fate. Based on in silico analyses of the important motifs present in the PDLP5 promoter combined with co-expression data, we present here a model illustrating what molecular networks PDLP5 might operate within during a defense response.

A computational investigation of conserved cis-elements present within the promoter region of PDLP5 revealed that it is enriched with stress-response sequences, as well as a W-box, a TGA-box and several as-1-like elements, binding sites for WRKY and TGA transcription factors, respectively (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (Fig. 1). WRKY factors are activated during a wide variety of stress-related events in plant cells, including SA defense and cell death.^2-4 Among known WRKY members, WRKY22, a dark-induced senescence regulator, was found to co-express with PDLP5 (Table 1), presenting a likelihood that this factor could be responsible for developmental induction of PDLP5 during senescence.⁴ TGA boxes and as-1-like elements are found in the promoters of many SA-responsive genes at both early (e.g., GST6) and late (e.g., PR1) time points.^5-7 Certain TGA members have been shown to interact with the downstream SA signaling factor NPR1.⁸ It will be interesting to see whether the presence of a TGA box is linked to NPR1-dependent expression of PDLP5; in fact, one possible candidate from the co-expression list, TGA1, interacts with NPR1 to function in defense via two cysteine residues reduced by SA-triggered redox changes.⁹

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Figure 1.

Predicted cis-elements in the PDLP5 promoter. W-boxes act as binding sites for WRKY type transcription factors, many of which are involved in cell death-related signaling in response to endogenous processes like senescence or defense reactions to invading pathogens. TGA-boxes and as-1-like elements are found in many pathogen stress-induced gene promoters, acting as binding sites for TGA transcription factors.

Consistent with the role for PDLP5 in SA-mediated immune responses, meta-examination of PDLP5 co-expression data (available at http://atted.jp/top_search.shtml) revealed an enrichment of GO terms related to defense responses, SA biosynthetic/signaling pathways, and senescence/cell death (Tables 1 and ​2).2). Among these, PAD4¹⁰ and ICS1¹¹ had been already shown to be required for full PDLP5 expression during defense-related responses.^1,12 However, given that the EDS1/PAD4 complex^13,14 also controls a positive SA-feedback pathway as a major upstream node in defense signaling,^10,15,16 and that chloroplastic ICS1 synthesizes 90% of the SA during a defense response,¹¹ the possibility cannot be excluded that these factors may also function downstream of PDLP5 in the positive feedback loop. Supporting this possible role for EDS1/PAD4 feedback is the fact that EDS1B (At3g48080), a functional homolog of EDS1 (At3g48090),¹⁷ is co-expressed with PDLP5. One appealing idea could be that this homolog is upregulated by PDLP5-induced SA accumulation to enhance the positive defense feedback loop initiated by EDS1.

Another interesting regulatory gene co-expressed with PDLP5 is PBS3, a cytosolic enzyme that conjugates amino acid groups to 4-substituted benzoates.^18,19 In triple mutants of ics1, pbs3, and the SA-overaccumulating mutant acd6 (acd6-1win3-1sid2-1), total SA levels are additively reduced, compared with double mutants acd6-1sid2-1 and acd6-1win3-1, demonstrating an important ICS1-independent role for PBS3 in SA production.²⁰ It would be exciting to find that PDLP5 enhances the SA feedback mechanism via both ICS1 and PBS3, which could lead to a maximum build-up of SA in a short amount of time. Conceivably, such molecular coordination across subcellular compartments would ensure that defense-related cell death progresses rapidly while remaining contained. Under this scenario, SA-stimulated PDLP5 accumulation at PD would help to prevent the symplastic leakage of any toxic molecules. The finding that PD closure is a direct response to pathogenic bacterial infection¹ also strengthens the argument that there could be endogenous or pathogenic non-cell-autonomous molecules that need to be blocked to alleviate, if not ameliorate, the effectiveness of an infection.

While the exact nature of the intercellular death-triggering signal(s) is currently not fully understood, reactive oxygen species (ROS) are possible candidates. A ROS burst is one hallmark of the HR, and ROS signaling has also been implicated in aspects of senescence and basal defense.^21-23 Cellular fate during a defense response is determined by the concentration and type of each ROS produced. ROS such as hydrogen peroxide may cause local cell death in high concentrations, but are also membrane-permeable and can pass quickly short distances into neighboring cells through the apoplast, eliciting downstream defense responses.²⁴ Other species like superoxide, are membrane impermeable, acting as a rapid initial signaling burst while also working together with other signals like NO and SA to induce cell death.^16,23,25,26 Cell-damaging toxic ROS, and other unknown signals that may accumulate to high concentrations during defense-related cell death, could threaten neighboring healthy cells if steps are not taken to reduce intercellular leakage. Thus, in order to confine these signals within the infected cells while still boosting immunity in neighboring cells through the action of short-distance signals like hydrogen peroxide, plant cells would need a mechanism utilizing both symplastic and apoplastic pathways. Here again, PDLP5 would be an excellent candidate for regulating symplastic permeability in response to changes in cellular redox condition. PDLP5 contains a cytoplasmic C-terminus relatively rich in cysteine residues, which might function as a redox-sensor similar to those in NPR1.²⁷ In fact, SA accumulation, which induces PDLP5 expression, also affects the redox balance.⁹ Not surprisingly, the list of genes co-expressed with PDLP5 contains many redox-regulators, including FMO1, a glutaredoxin, and a subunit of NADPH respiratory burst oxidase (Table 1 and ​2).2). Understanding how these potential redox components might function in conjunction with PDLP5 and/or influence PDLP5 activity would be an insightful future endeavor.

By incorporating the circumstantial evidence described above into the data known so far about PDLP5, we propose a theoretical model illustrating a potential network supporting the PDLP5 function in regulating non-cell-autonomous cell death signals (Fig. 2). Upon pathogen recognition, plant cells respond by altering the cellular redox environment and activating EDS1/PAD4 control of SA accumulation. This turns on defense-related transcription factors like NPR1/TGA and WRKYs, which lead to the expression of downstream genes including PDLP5. Accumulation of PDLP5 at PD potentiates redox modifications via positive SA feedback. The buildup of SA and ROS triggers cell death in the immediate vicinity of the infection, but the symplastic spread of death-inducing toxins is limited by the action of PDLP5 at the PD. The local effect of PDLP5 could be multifaceted, recruiting callose and partially occluding PD while simultaneously triggering an SA biosynthesis/modification/regulatory circuit. This model represents our best interpretation of PDLP5 function and highlight important questions. For example, what are the molecular players and mechanisms responsible for PDLP5-induced PD callose deposition? What is the direct target of PDLP5 that turns on the SA amplification pathway? Does ROS or redox status have direct impact on PDLP5 function, or vice versa? Certainly, answering these questions would help to comprehend the complexity and significance of the role that PD play to bring about supracellular defense control in plants.

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Figure 2.

A schematic illustration depicting potential defense signaling cascades and networks across PD. Microbial pathogens are recognized by pathogen recognition receptors (PRRs) at the cell surface, initiating a mitogen-activated protein kinase (MAPK) cascade that signals plasma membrane-localized NADPH oxidase (Atrobh) to produce a burst of reactive oxygen species like superoxide (O₂-), which through dismutation can become hydrogen peroxide (H₂O₂). The ROS burst triggers EDS1 and PAD4 to form a complex that moves to the nucleus, leading to expression of salicylic acid biosynthetic genes like ICS1. Cytoplasmic SA accumulation and redox change releases NPR1 from its oligomeric form, allowing it to move to the nucleus and bind to defense-related TGA transcription factors. These factors, as well as others like WRKYs, induce PDLP5 expression along with other defense genes. As a type I transmembrane protein, PDLP5 is targeted to PD via a secretory system. At PD, PDLP5 reduces the size exclusion limit by recruiting callose and/or by occluding the cytoplasmic sleeves as oligomeric complex formed through intermolecular disulfide-linkages. PDLP5 also functions in a feedback loop that reinforces SA accumulation (possibly through PBS3), contributing to cell death; however, its action at PD helps to block the symplastic spread of any harmful cell death-related signals into neighboring healthy cells. These uninfected cells, which have been primed by the apoplastic movement of membrane-permeable ROS signals like H₂O₂, upregulate SA and redox changes but only to an extent that activates basal immunity, not cell death. PDLP5 expression and localization in these cells further prevents influx of harmful compounds by restricting PD.

Acknowledgments

Notes

Lee J-Y, Wang X, Cui W, Sager R, Modla S, Czymmek K, et al. A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis Plant Cell 2011 23 3353 73 doi: 10.1105/tpc.111.087742.

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