The processed C-terminus of AvrRps4 effector suppresses plant immunity via targeting multiple WRKYs.

Pathogens generate and secrete effector proteins to the host plant cells during pathogenesis to promote virulence and colonization. If the plant carries resistance (R) proteins that recognize pathogen effectors, effector-triggered immunity (ETI) is activated, resulting in a robust immune response and hypersensitive response (HR). The bipartite effector AvrRps4 from Pseudomonas syringae pv. pisi has been well studied in terms of avirulence function. In planta, AvrRps4 is processed into two parts. The C-terminal fragment of AvrRps4 (AvrRps4C) induces HR in turnip and is recognized by the paired resistance proteins AtRRS1/AtRPS4 in Arabidopsis. Here, we show that AvrRps4C targets a group of Arabidopsis WRKY, including WRKY46, WRKY53, WRKY54, and WRKY70, to induce its virulence function. Indeed, AvrRps4C suppresses the general binding and transcriptional activities of immune-positive regulator WRKY54 and WRKY54-mediated resistance. AvrRps4C interferes with WRKY54's binding activity to target gene SARD1 in vitro, suggesting WRKY54 is sequestered from the SARD1 promoter by AvrRps4C. Through the interaction of AvrRps4C with four WRKYs, AvrRps4 enhances the formation of homo-/heterotypic complexes of four WRKYs and sequesters them in the cytoplasm, thus inhibiting their function in plant immunity. Together, our results provide a detailed virulence mechanism of AvrRps4 through its C-terminus.

Engineering plant immune circuit: walking to the bright future with a novel toolbox.

Plant pathogens destroy crops and cause severe yield losses, leading to an insufficient food supply to sustain the human population. Apart from relying on natural plant immune systems to combat biological agents or waiting for the appropriate evolutionary steps to occur over time, researchers are currently seeking new breakthrough methods to boost disease resistance in plants through genetic engineering. Here, we summarize the past two decades of research in disease resistance engineering against an assortment of pathogens through modifying the plant immune components (internal and external) with several biotechnological techniques. We also discuss potential strategies and provide perspectives on engineering plant immune systems for enhanced pathogen resistance and plant fitness.

Pathogen effectors: What do they do at plasmodesmata?

Plants perceive an assortment of external cues during their life cycle, including abiotic and biotic stressors. Biotic stress from a variety of pathogens, including viruses, oomycetes, fungi, and bacteria, is considered to be a substantial factor hindering plant growth and development. To hijack the host cell's defence machinery, plant pathogens have evolved sophisticated attack strategies mediated by numerous effector proteins. Several studies have indicated that plasmodesmata (PD), symplasmic pores that facilitate cell-to-cell communication between a cell and neighbouring cells, are one of the targets of pathogen effectors. However, in contrast to plant-pathogenic viruses, reports of fungal- and bacterial-encoded effectors that localize to and exploit PD are limited. Surprisingly, a recent study of PD-associated bacterial effectors has shown that a number of bacterial effectors undergo cell-to-cell movement via PD. Here we summarize and highlight recent advances in the study of PD-associated fungal/oomycete/bacterial effectors. We also discuss how pathogen effectors interfere with host defence mechanisms in the context of PD regulation.

Nuclear Localization of HopA1Pss61 Is Required for Effector-Triggered Immunity.

Plant resistance proteins recognize cognate pathogen avirulence proteins (also named effectors) to implement the innate immune responses called effector-triggered immunity. Previously, we reported that hopA1 from Pseudomonas syringae pv. syringae strain 61 was identified as an avr gene for Arabidopsis thaliana. Using a forward genetic screen approach, we cloned a hopA1-specific TIR-NBS-LRR class disease resistance gene, RESISTANCE TO PSEUDOMONAS SYRINGAE6 (RPS6). Many resistance proteins indirectly recognize effectors, and RPS6 is thought to interact with HopA1Pss61 indirectly by surveillance of an effector target. However, the involved target protein is currently unknown. Here, we show RPS6 is the only R protein that recognizes HopA1Pss61 in Arabidopsis wild-type Col-0 accession. Both RPS6 and HopA1Pss61 are co-localized to the nucleus and cytoplasm. HopA1Pss61 is also distributed in plasma membrane and plasmodesmata. Interestingly, nuclear localization of HopA1Pss61 is required to induce cell death as NES-HopA1Pss61 suppresses the level of cell death in Nicotiana benthamiana. In addition, in planta expression of hopA1Pss61 led to defense responses, such as a dwarf morphology, a cell death response, inhibition of bacterial growth, and increased accumulation of defense marker proteins in transgenic Arabidopsis. Functional characterization of HopA1Pss61 and RPS6 will provide an important piece of the ETI puzzle.

Near-IR and CP-OCT imaging of suspected occlusal caries lesions.

INTRODUCTION: Radiographic methods have poor sensitivity for occlusal lesions and by the time the lesions are radiolucent they have typically progressed deep into the dentin. New more sensitive imaging methods are needed to detect occlusal lesions. In this study, cross-polarization optical coherence tomography (CP-OCT) and near-IR imaging were used to image questionable occlusal lesions (QOC's) that were not visible on radiographs but had been scheduled for restoration on 30 test subjects. METHODS: Near-IR reflectance and transillumination probes incorporating a high definition InGaAs camera and near-IR broadband light sources were used to acquire images of the lesions before restoration. The reflectance probe utilized cross-polarization and operated at wavelengths from 1,500 to 1,700 nm where there is an increase in water absorption for higher contrast. The transillumination probe was operated at 1,300 nm where the transparency of enamel is highest. Tomographic images (6 × 6 × 7 mm3 ) of the lesions were acquired using a high-speed swept-source CP-OCT system operating at 1,300 nm before and after removal of the suspected lesion. RESULTS: Near-IR reflectance imaging at 1,500-1,700 nm yielded significantly higher contrast (P < 0.05) of the demineralization in the occlusal grooves compared with visible reflectance imaging. Stains in the occlusal grooves greatly reduced the lesion contrast in the visible range yielding negative values. Only half of the 26 lesions analyzed showed the characteristic surface demineralization and increased reflectivity below the dentinal-enamel junction (DEJ) in 3D OCT images indicative of penetration of the lesion into the dentin. CONCLUSION: This study demonstrates that near-IR imaging methods have great potential for improving the early diagnosis of occlusal lesions. Lasers Surg. Med. 49:215-224, 2017. © 2017 Wiley Periodicals, Inc.

Enamel Thickness Determination by Optical Coherence Tomography: In vitro Validation.

Optical coherence tomography (OCT) has been introduced in dentistry as a nondestructive diagnostic imaging tool that does not utilize ionizing radiation. This study investigated the agreement between polarization-sensitive OCT (PS-OCT), microcomputed tomography (µ-CT), and histology for enamel thickness measurements. Human enamel samples were prepared and evaluated with µ-CT and PS-OCT and then sectioned and observed via digital transversal light microscopy. For all methods, a standard transversal section (b-scan) in each sample was selected, and the enamel thickness was measured at three predetermined positions using ImageJ analysis software. The results revealed significant high agreement between all tested methods, indicating the potential of PS-OCT as a clinical tool to effectively measure enamel thickness.

Use of an optical clearing agent to enhance the visibility of subsurface structures and lesions from tooth occlusal surfaces.

The purpose of this study is to show that optical clearing agents can be used to increase the visibility of deeply penetrating occlusal lesions that have reached the underlying dentin and spread laterally under the enamel. Previous studies have shown that high refractive index fluids can increase the contrast of caries lesions. Extracted teeth with natural occlusal lesions were imaged with optical coherence tomography (OCT) with and without the addition of a transparent vinyl polysiloxane impression material (VPS) currently used in vivo. The relative intensity of the reflectivity from the underlying lesion area for each sample was measured before and after application of the VPS. Lesion presence was confirmed with polarized light microscopy and microradiography. Application of VPS significantly increased (P < 0.0001) the integrated reflectivity of subsurface dentinal lesions. This study shows that optical clearing agents can be used to increase the optical penetration and the visibility of subsurface lesions and the dentinal­enamel junction under sound and demineralized enamel in OCT images.

Enhancement of OCT images with vinyl polysiloxane (VPS).

Several studies have shown that optical coherence tomography (OCT) can be used to measure the remaining enamel thickness and detect the location of subsurface lesions hidden under the sound enamel. Moreover studies have shown that high refractive index liquids can be used to improve the visibility of subsurface lesions in OCT images. In this study, we demonstrate that vinyl polysiloxane (VPS) impression materials which are routinely used in dentistry can be used to enhance the detection of dentinal lesions on tooth occlusal surfaces. Lesion presence was confirmed with polarized light microscopy and microradiography.

Clinical monitoring of smooth surface enamel lesions using CP-OCT during nonsurgical intervention.

INTRODUCTION: Studies have shown that cross-polarization optical coherence tomography (CP-OCT) can be used to image the internal structure of carious lesions in vivo. The objective of this study was to show that CP-OCT can be used to monitor changes in the internal structure of early active carious lesions on smooth surfaces during non-surgical intervention with fluoride. METHODS: Lesions on the smooth surfaces of teeth were imaged using CP-OCT on 17 test subjects. Lesion structural changes were monitored during fluoride varnish application at 6-week intervals for 30 weeks. The lesion depth (Ld ), integrated reflectivity (ΔR), and surface zone thickness (Sz ) were monitored. RESULTS: A distinct transparent surface zone that may be indicative of lesion arrestment was visible in CP-OCT images on 62/63 lesions before application of fluoride varnish. The lesion depth and internal structure were resolved for all the lesions. The overall change in the mean values for Ld , ΔR, and Sz for all the lesions was minimal and was not significant during the study (P > 0.05). Only 5/63 lesions manifested a significant increase in Sz during intervention. CONCLUSION: Even though it appears that most of the lesions manifested little change with fluoride varnish application in the 30 weeks of the study, CP-OCT was able to measure the depth and internal structure of all the lesions including the thickness of the important transparent surface zone located at the surface of the lesions, indicating that CP-OCT is ideally suited for monitoring lesion severity in vivo. Lasers Surg. Med. 48:915-923, 2016. © 2016 Wiley Periodicals, Inc.

Enhanced detection of dentinal lesions in OCT images using the RKT transformation.

Several studies have shown that optical coherence tomography (OCT) can be used to measure the remaining enamel thickness and detect the location of subsurface lesions hidden under the sound enamel. The purpose of this study was to develop algorithms to enhance the visibility of subsurface structures such as hidden occlusal lesions and the dentinal-enamel junction. Extracted teeth with natural occlusal lesions were imaged with OCT with and without added high index fluids. A Rotating Kernel Transformation (RKT) nonlinear image processing filter was applied to PS-OCT images to enhance the visibility of the subsurface lesions under the sound enamel. The filter significantly increased (P<0.05) the visibility of the subsurface lesions.

Automated assessment of the remineralization of artificial enamel lesions with polarization-sensitive optical coherence tomography.

Accurate measurement of the highly mineralized transparent surface layer that forms on caries lesions is important for diagnosis of the lesion activity because chemical intervention can slow or reverse the caries process via remineralization. Previous in-vitro and in-vivo studies have demonstrated that polarization-sensitive optical coherence tomography (PS-OCT) can nondestructively image the subsurface lesion structure and the highly mineralized transparent surface zone of caries lesions. The purpose of this study was to develop an approach to automatically process 3-dimensional PS-OCT images and to accurately assess the remineralization process in simulated enamel lesions. Artificial enamel lesions were prepared on twenty bovine enamel blocks using two models to produce varying degree of demineralization and remineralization. The thickness of the transparent surface layer and the integrated reflectivity of the subsurface lesion were measured using PS-OCT. The automated transparent surface layer detection algorithm was able to successfully detect the transparent surface layers with high sensitivity ( = 0.92) and high specificity ( = 0.97). The estimated thickness of the transparent surface layer showed a strong correlation with polarized light microscopy (PLM) measurements of all regions (R(2) = 0.90). The integrated reflectivity, ΔR, and the integrated mineral loss, ΔZ, showed a moderate correlation (R(2) = 0.32). This study demonstrates that PS-OCT can automatically measure the changes in artificial enamel lesion structure and severity upon exposure to remineralization solutions.

Enhancing the detection of hidden occlusal caries lesions with OCT using high index liquids.

In a previous study, we investigated the influence of several high refractive index fluids on the performance of polarization sensitive optical coherence tomography (PS-OCT). That study showed that these liquids can increase the effective imaging depth and lesion contrast. Other in vitro and in vivo studies have shown that OCT can be used to show whether occlusal lesions have penetrated to the dentinal-enamel junction (DEJ) and spread laterally under the enamel. The purpose of this study was to determine if high index fluids can enhance the ability of OCT to detect hidden occlusal lesions and show if these lesions have penetrated through the enamel into the underlying dentin. Ten extracted teeth with occlusal lesions were imaged using OCT after the application of water, glycerol, BABB (33% Benzyl Alcohol + 67% Benzyl Benzoate) and a Cargille Liquid (Cedar Grove, NJ) (hydrogenated terphenyl 1-bromo-naphthalene) with a refractive index of 1.61. The intensity of the reflectance from the underlying lesion area for each sample was compared with the reflectance of the sound tooth surface for each fluid. The use of high index fluids significantly (P< 0.0001) increased the reflectivity of subsurface occlusal lesions under the surrounding sound enamel.

Longitudinal monitoring of demineralization peripheral to orthodontic brackets using cross polarization optical coherence tomography.

OBJECTIVES: The aim of this study was to test the hypothesis that cross-polarization optical coherence tomography (CP-OCT) can be used to longitudinally monitor demineralization peripheral to orthodontic brackets in an extended clinical study. METHODS: A high-speed CP-OCT system was used to acquire 3D volumetric images of the area at the base of orthodontic brackets over a period of 12 months after placement. The reflectivity was measured at 3-month intervals for 12 months to determine if there was increased demineralization. Two teeth were monitored on 20 test subjects and the brackets were bonded using two types of adhesives. This was a randomized controlled clinical study with a split mouth design such that each subject served as his or her own control. On one side, the control premolar was bonded with a bonding agent (Adper Scotchbond from 3M ESPE, St. Paul, MN) and composite (Transbond XT from 3M Unitek, Monrovia, CA) that lacked fluoride. On the other side, the experimental premolar was bonded with a fluoride releasing glass ionomer cement (GC Fuji Ortho LC from GC America, Alsip, IL). RESULTS: There was a small but significant increase in the calculated lesion depth and integrated reflectivity over that depth (ΔR) for both adhesive types (p<0.0001) indicating increasing demineralization with time. There was no significant difference in the lesion depth (p=0.22) and ΔR (p=0.91) between the groups with the fluoride releasing glass ionomer cement and the conventional composite. CONCLUSIONS: CP-OCT was able to measure a significant increase in demineralization (p<0.0001) at the base of orthodontic brackets over a period of 12 months.

Monitoring the Remineralization of Early Simulated Lesions using a pH Cycling Model with CP-OCT.

If caries lesions are detected early enough they can be arrested by chemical intervention and dietary changes without the need for chemical intervention. Optical coherence tomography is ideally suited to monitor the changes that occur in caries lesions as a result of nonsurgical intervention, since OCT can nondestructively image the internal structure of the lesion. One of the most important changes that occurs in a lesion is preferential deposition of mineral in the outer surface zone. The deposition creates a highly mineralized and weakly scattering surface zone that is clearly visible in OCT images. Since this zone is near the highly reflective surface it is necessary to use cross-polarization OCT imaging to resolve this zone. Several CP-OCT studies have been conducted employing different remineralization models that produce lesions with varying mineral gradients. Previous studies have also demonstrated that automated algorithms can be used to assess the lesion depth and severity even with the presence of the weakly reflective surface zone. In this study we investigated the remineralization of lesions of varying severity using a pH cycling remineralization model and the change of the lesion was monitored using CP-OCT. Although the lesion depth and severity decreased after remineralization, there was still incomplete remineralization of the body of the lesion.

The valosin-containing protein promotes cardiac survival through the inducible isoform of nitric oxide synthase.

AIMS: Expression of the heat shock protein 22 (Hsp22) in the heart stimulates cardiac cell survival through activation of the Akt pathway and expression of the inducible nitric oxide (NO) synthase (iNOS), the mediator of ischaemic preconditioning and the most powerful prophylaxis against cardiac cell death. The goal of the present study was to elucidate the downstream effector by which Hsp22 and Akt increase iNOS expression. We tested both in vivo and in vitro the hypothesis that such an effector is the valosin-containing protein (VCP), an Akt substrate, which activates the transcription factor NF-κB, using a transgenic mouse with cardiac-specific over-expression of Hsp22, as well as isolated rat cardiac myocytes. METHODS AND RESULTS: Using two-dimensional gel electrophoresis and mass spectrometry combined with immunoprecipitation, we found that Hsp22 and Akt co-localize and interact together with VCP. Adeno-mediated over-expression of VCP in isolated cardiac myocytes activated NF-κB and dose-dependently increased the expression of iNOS, which was abolished upon NF-κB inhibition. Over-expression of a dominant-negative (DN) mutant of VCP did not increase iNOS expression. VCP, but not its DN mutant, protected against chelerythrine-induced apoptosis, which was suppressed by inhibition of either NF-κB or iNOS. VCP-mediated activation of the NF-κB/iNOS pathway was also prevented upon inhibition of Akt. CONCLUSION: We conclude that the Akt substrate, VCP, mediates the increased expression of iNOS downstream from Hsp22 through an NF-κB-dependent mechanism.

Monitoring tooth demineralization using a cross polarization optical coherence tomographic system with an integrated MEMS scanner.

New methods are needed for the nondestructive measurement of tooth demineralization and remineralization to monitor the progression of incipient caries lesions (tooth decay) for effective nonsurgical intervention and to evaluate the performance of anti-caries treatments such as chemical treatments or laser irradiation. Studies have shown that optical coherence tomography (OCT) has great potential to fulfill this role since it can be used to measure the depth and severity of early lesions with an axial resolution exceeding 10-µm, it is easy to apply in vivo and it can be used to image the convoluted topography of tooth occlusal surfaces. In this paper we present early results using a new cross-polarization OCT system introduced by Santec. This system utilizes a swept laser source and a MEMS scanner for rapid acquisition of cross polarization images. Preliminary studies show that this system is useful for measurement of the severity of demineralization on tooth surfaces and for showing the spread of occlusal lesions under the dentinal-enamel junction.

Remineralization of Root Caries Monitored Using Cross-Polarization Optical Coherence Tomography.

Previous studies have demonstrated that polarization sensitive optical coherence tomography (PS-OCT) can be used to image caries lesions in dentin, measure nondestructively the severity of dentin demineralization, and determine the efficacy of intervention with anticaries agents including fluoride and lasers. However, those studies were limited to artificial lesions on dentin and roots surfaces. The objective of this study is to determine if a cross polarization OCT system (CP-OCT) can be used to nondestructively measure a reduction in the reflectivity of natural root caries lesions after exposure to a remineralization solution. CP-OCT images of 11 teeth with existing root lesions were acquired before and after exposure to a remineralizing solution for 20 days. The integrated reflectivity was calculated after integrating to a fixed depth of 200-µm. There was a significant decrease in the integrated reflectivity after exposure to the remineralizing solution.

Nondestructive Monitoring of the Repair of Natural Occlusal Lesions using Cross - Polarization Optical Coherence Tomography.

Previous remineralization studies employing cross polarization sensitive optical coherence tomography (CP-OCT), have been limited to the repair of artificial enamel-like lesions. In this study we attempted to remineralize existing occlusal lesions on extracted teeth. Lesions were imaged before and after exposure to an acidic remineralization regimen and the integrated reflectivity and lesion depth was calculated. Automated integration routines worked well for assessing the integrated reflectivity for the lesion areas after remineralization. Polarized light microscopy was also used to examine the lesions areas after sectioning the teeth. An acidic remineralization solution was used to remineralize the lesions. The integrated reflectivity significantly increased after exposure to the remineralization solution which suggests that the acidic solution caused additional demineralization as opposed to the desired remineralization.

Investigation of Acid-Etched CO2 Laser Ablated Enamel Surfaces Using Polarization Sensitive Optical Coherence Tomography.

A carbon dioxide laser operating at the highly absorbed wavelength of 9.3µm with a pulse duration of 10-15µs is ideally suited for caries removal and caries prevention. The enamel thermally modified by the laser has enhanced resistance to acid dissolution. This is an obvious advantage for caries prevention; however, it is often necessary to etch the enamel surface to increase adhesion to composite restorative materials and such surfaces may be more resistant to etching. The purpose of the study was to non-destructively measure the susceptibility of laser-ablated enamel surfaces to acid dissolution before and after acid-etching using Polarization Sensitive Optical Coherence Tomography (PS-OCT). PS-OCT was used to acquire images of bovine enamel surfaces after exposure to laser irradiation at ablative fluence, acid-etching, and a surface softened dissolution model. The integrated reflectivity from lesion and the lesion depth were measured using PS-OCT. Samples were also sectioned for examination by Polarized Light Microscopy (PLM). PS-OCT images showed that acid-etching greatly accelerated the formation of subsurface lesions on both laser-irradiated and non-irradiated surfaces (P<0.05). A 37.5% phosphoric acid etch removed the laser modified enamel layer after 5-10 seconds.

Nondestructive monitoring of the repair of enamel artificial lesions by an acidic remineralization model using polarization-sensitive optical coherence tomography.

OBJECTIVES: It is difficult to completely remineralize carious lesions because diffusion into the interior of the lesion is inhibited as new mineral is deposited in the outermost layers. In previous remineralization studies employing polarization sensitive optical coherence tomography (PS-OCT), two models of remineralization were employed and in both models there was preferential deposition of mineral in the outer most layer. In this study we attempted to remineralize the entire lesion using an acidic remineralization model and demonstrate that this remineralization can be monitored using PS-OCT. METHODS: Artificial lesions approximately 100-150 µm in-depth were exposed to an acidic remineralization regimen and the integrated reflectivity from the lesions was measured before and after remineralization using PS-OCT. RESULTS: Automated integration routines worked well for assessing the integrated reflectivity for the lesion areas after remineralization. Although there was a high degree of remineralization, there was still incomplete remineralization of the body of the lesion. CONCLUSION: This study demonstrated that PS-OCT can be used to non-destructively measure changes in lesion structure and severity upon exposure to an acidic remineralization model. This study also demonstrated that automated algorithms can be used to assess the lesion severity even with the presence of a weakly reflective surface zone.