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Lesion gaps assessed by late-gadolinium enhancement magnetic resonance imaging (LGE-MRI) are associated with the atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI). Animal studies have demonstrated that the catheter-contact force (CF), stability, and orientation are strongly associated with lesion formation. However, the impact of those procedural factors on the lesion characteristics associated with AF recurrence has not been well discussed.A total of 30 patients with paroxysmal AF who underwent catheter ablation were retrospectively enrolled. Radiofrequency (RF) applications were performed with 35W for 30s in a point-by-point fashion under esophageal temperature monitoring. The inter-lesion distance was 4mm. The lesions were visualized by LGE-MRI three months post-procedure and assessed by the LGE volume (ml), gap number (GN), and average gap length (AGL [mm]). The gaps were defined as non-enhancement sites of4 mm. The procedural factors including the catheter-CF, stability, and orientation were calculated on the NavX system.Six (20%) of 30 patients had AF recurrences 12 months post-ablation. A univariate analysis demonstrated that the AGL was associated with AF recurrence (hazard ratio [HR]: 1.20, confidence interval [CI]:1.03 - 1.42, p = 0.02). All AF recurrence were found in patients with an AGL of7 mm. The catheter-CF and stability were associated with an AGL of7mm, but not the orientation (CF: HR: 0.62, CI: 0.39-0.97, p=0.038; stability: HR: 0.8, CI: 0.66-0.98, p=0.027).RF ablation with a low CF and poor catheter stability has a potential risk of creating large lesion gaps associated with AF recurrence. This article is protected by copyright. All rights reserved.

Background: The mechanism underlying the sex differences in atrial fibrillation (AF) recurrence following pulmonary vein (PV) isolation is not fully understood. We hypothesized that non-PV foci and epicardial adipose tissue (EAT) play a key role. Methods and Results: Data from 304 consecutive patients (75% males) who underwent contrast-enhanced computed tomography and catheter ablation of AF were reviewed. The EAT around the atrium was measured separately in 4 parts of the atrium. All patients underwent high-dose isoproterenol infusions to assess the non-PV foci. Significantly more non-PV foci and less EAT around the atrium were observed in female patients than in male patients. In males, those with non-PV foci on the left atrial (LA) anterior wall had significantly greater EAT for the same lesions than those without non-PV foci. During a median follow-up of 27 months, the predictors of AF recurrence after first catheter ablation were female sex, presence of non-PV foci, LA diameter, and septal EAT index. A sex-specific analysis revealed that LA diameter was a predictor only in males and that the presence of non-PV foci in the septal region was a strong predictor in males (hazard ratio [HR]: 2.24) and females (HR: 3.65). Conclusions: Sex-specific differences were observed in non-PV foci sites and local EAT and in regard to the predictors of AF recurrence.

Background: Ventricular tachycardia (VT) non-inducibility at the end of ablation is associated with a less likely VT recurrence. However, it is not clear whether we should use VT non-inducibility as a routine end point of VT ablation. The aim of this study was to evaluate VT recurrence in patients in whom VT non-inducibility was not achieved at the end of the RF ablation and the factors attributing to the VT recurrence.Methods: We analyzed 62 patients in whom VT non-inducibility was not achieved at the end of the RF ablation were studied.Results: Over 2 years, 22 (35%) of the cases had VT recurrences. A multivariate analysis showed that an LVEF≥35% (HR:0.19; 95% CI:0.06- 0.49; PConclusion: Even if VT non-inducibility was not achieved, patients with an LVEF≥35% or in whom partial success could be achieved might be prevented from having VT recurrences. The validity of the VT non-inducibility of any VT should be evaluated considering each patient’s background and the results of the procedure.

The skin overlying cardiovascular implantable electronic devices (CIEDs) sometimes becomes very thin after implantations, which could cause a device erosion. The factors related to the skin thickness of device pockets have not been elucidated. This study aimed to evaluate the skin thickness of CIED pockets and search for the factors associated with the thickness.Seventeen skin thickness points around the CIED pocket were measured through ultrasonography in each patient.A total of 101 patients (76 ± 11 years, 26 female) were enrolled. The median duration from the implantation to the examination was 95 months (quartile: 52.5-147.5). The median skin thickness overlying the device was 4.1 mm (3.3-5.9). Patients with heart failure and malignancy had thinner skin overlying the CIED than those without. A significant correlation existed between skin thickness and body mass index (BMI), hemoglobin, serum creatinine, estimated glomerular filtration rate (eGFR), and left ventricular ejection fraction. In contrast, age, gender, and device size did not exhibit a significant correlation with skin thickness. A multivariate logistic regression analysis revealed that chronic heart failure and a decrease in the eGFR and BMI were independent predictive factors of "very thin (≦3.3 mm)" skin of the CIED pocket late after an implantation.Aside from a low BMI, the comorbidities (low hemoglobin, heart failure, and renal dysfunction) had a stronger impact on the skin thickness overlying the device than the device size. A careful observation of the device pocket should be performed in patients with those risk factors.

Eosinophilic myocarditis (EM) is an under-diagnosed inflammatory heart disease that often leads to severe left ventricular (LV) dysfunction. Meanwhile, severe secondary mitral regurgitation (MR) with valve disruption, possibly requiring mitral valve repair, is rarely concomitant with EM. We present the case of a 64-year-old female diagnosed with heart failure with severe LV dysfunction and localized asynergy. Echocardiography revealed severe secondary MR with mitral valve disruption. Cardiac magnetic resonance imaging (CMR) showed transmural late-gadolinium enhancement localized in the anterior wall and diffuse high-signal areas on T2-weighted images, suggesting non-ischemic and inflammatory heart disease. Although the peripheral eosinophil count was not elevated on admission, it gradually increased during hospitalization. These findings encouraged us to perform endomyocardial biopsy, which confirmed myocardial eosinophilic infiltration with mild fibrosis and necrosis, leading to the diagnosis of EM. Immunosuppressive treatment with oral corticosteroids improved LV dysfunction and completely resolved severe secondary MR. The current case highlighted that comprehensive assessment of laboratory, imaging, and pathological examinations including CMR is crucial to develop the appropriate therapeutic strategy for refractory heart failure. Immunosuppressive treatment should be considered as the first therapeutic option even in EM cases with severe secondary MR, possibly requiring mitral valve repair. Learning objective The gradual increase in peripheral eosinophils during hospitalization, without significant peripheral eosinophilia on admission, is crucial for the diagnosis of eosinophilic myocarditis. Comprehensive assessment of laboratory, imaging, and pathological examinations including cardiac magnetic resonance imaging is mandatory when building an appropriate therapeutic strategy for refractory heart failure. Severe secondary mitral regurgitation with mitral valve disruption can be completely resolved via immunosuppressive treatment in cases of eosinophilic myocarditis.

International audience; ChEC91, a novel cell death-inducing effector protein from the fungal pathogen Colletotrichum higginsianum, causal agent of crucifer anthracnose disease, is described. Both transient expression of ChEC91 and infiltration of purified recombinant protein induced necrotic lesions in Nicotiana benthamiana leaves. The recombinant protein also induced electrolyte leakage and callose deposition in Arabidopsis thaliana leaf tissue and the expression of defence marker genes. Moreover, fungal mutants constitutively over-expressing ChEC91 in C. higginsianum were impaired in appressorial penetration on Brassica rapa cotyledons. These results suggest that inappropriate expression of ChEC91 might negatively affect the early stage of C. higginsianum infection by inducing plant defence responses. Protein domain deletion analysis showed that the C-terminal region of ChEC91 was necessary, but not sufficient, for activity in N. benthamiana. Homologous effector proteins cloned from C. gloeosporioides, Fusarium graminearum, and Pyricularia oryzae differed in their cell death-inducing activity, which appeared related to sequence variations in the C-terminal region of these proteins. Moreover, this region contained amino acid residues that were well conserved within Colletotrichum species. These results suggest that the amino acid residues in the C-terminal region may be important for inducing cell death in plants.

Background: Demand is growing for remote electrocardiogram (ECG) monitoring systems in the COVID-19 era in Japan. This study describes initial experiences with a small wireless ECG monitoring device and the utility of delivery via the postal service for outpatient care in Japan. Methods and Results: Long-term ECG monitoring following postal delivery of the small ECG device was evaluated in 25 patients. The patients had no difficulties with either the postal delivery or self-fitting and wearing the devices. A median of 57 h monitoring per patient was performed. Arrhythmic events were detected in 8 patients. Most patients were satisfied with both the ECG devices and postal delivery. Conclusions: Postal delivery of ECG devices could be used in clinical practice to achieve less or no in-person contact during the COVID-19 era.

Background A computational model demonstrated that atrial fibrillation (AF) rotors could be distributed in patchy late-gadolinium enhancement (LGE) areas and play an important role in AF drivers. However, this was not validated in humans. Objective The purpose of this study was to evaluate the LGE area properties of AF rotors in patients with persistent AF. Methods A total of 287 segments in 15 patients with persistent AF (longstanding persistent AF in 9 patients) that underwent AF ablation were assessed. Non-passively activated areas (NPAs), where rotational activation (AF rotor) was frequently observed, were detected by the novel real-time phase mapping (ExTRa Mapping). The properties of the LGE areas were assessed using the LGE heterogeneity, and the density was assessed by the entropy (LGE-entropy) and volume ratio of the enhancement voxel (LGE-volume ratio), respectively. Results NPAs were found in 61 (21%) of 287 segments and were mostly found around the pulmonary vein antrum. A receiver operating characteristic curve analysis yielded an optimal cutoff value of 5.7 and 10% for the LGE-entropy and LGE-volume ratio, respectively. The incidence of NPAs was significantly higher at segments with an LGE-entropy of >5.7 and LGE-volume ratio of >10% than at the other segments (38 [30%] of 126 vs. 23 [14%] of 161 segments, p = 0.001). No NPAs were found at segments with an LGE-volume ratio of >50% regardless of the LGE-entropy. Of 5 patients with AF recurrence, NPAs outside the PV antrum were not ablated in 3 patients and the remaining NPAs were ablated, but their LGE-entropy and LGE-volume ratio were low. Conclusion AF rotors are mostly distributed in relatively weak and much more heterogenous LGE areas. This article is protected by copyright. All rights reserved.

Background Inflammation, such as that associated with intermediate CD14++ CD16+ monocytes and atrial structural remodeling (SRM), may be important in the recurrence of atrial fibrillation (AF) after catheter ablation. However, the relationship between the intermediate CD14++ CD16+ monocytes, SRM, and AF recurrence is unclear. Methods Twenty-four patients with AF were enrolled. The proportion of intermediate monocytes (PIM) was assessed before ablation by flow cytometry. As a surrogate marker of SRM, the volume ratio (VR) of signal intensity greater than 1 standard deviation on late-gadolinium enhancement magnetic resonance imaging (LGE-MRI) was calculated. We investigated whether PIM correlated with SRM on LGE-MRI and determined the optimal cutoff value for predicting AF recurrence. Results Univariate analysis revealed positive correlations between PIM and BNP with SRM (PIM: r = .593, p = .002; BNP: r = .567, p = .004). Multivariable analysis revealed that PIM was independently associated with VR on LGE-MRI (β = .522; p = .033). The finding of an area under the receiver operating characteristic curve of 0.750 revealed that a VR ≥ 13.3% on LGE-MRI as the optimal cutoff value to predict AF recurrence with 80% sensitivity and 71% specificity, which was associated with PIM ≥ 10.0%. Conclusion Intermediate monocytes were significantly positively correlated with SRM. PIM ≥ 10% was associated with a VR ≥ 13.3% on LGE-MRI, which predicted AF recurrence after catheter ablation.

BACKGROUND: A computer simulation model has demonstrated that an atrial fibrillation (AF) driver can be attached to heterogeneous fibrosis assessed by late gadolinium enhancement magnetic resonance imaging (LGE-MRI). However, it has not been well elucidated in patients with persistent AF. The aim of this study was to investigate whether radiofrequency (RF) applications in the fragmented LGE area (FLA) could terminate AF or convert it to atrial tachycardia (AT) and improve the rhythm outcome. METHODS: A total of 31 consecutive persistent AF patients with FLAs were enrolled (FLA ablation group, mean age: 69 ± 8 years, mean left atrial diameter: 42 ± 6 mm). A favorable response was defined as direct AF termination or AT conversion during RF applications at the FLA. The rhythm outcome was compared between the FLA ablation group and FLA burden-matched pulmonary vein isolation (PVI) group. RESULTS: Favorable responses were found in 15 (48%) of 31 patients in the FLA group (AF termination in 7, AT conversion in 8 patients), but not in the PVI group. AF recurrence at 12 months follow-up was significantly less in the FLA ablation group than in the PVI group (4 [13%] vs. 12 [39%] of 31 patients, log-rank p=0.023). In patients with a favorable response, AT recurred in 1 (7%) of 15 patients but AF did not. CONCLUSIONS: FLA ablation could terminate AF or convert it to AT in half of the patients. No AF recurrence was documented in patients with a favorable response. This article is protected by copyright. All rights reserved.

Background: Several algorithms have been proposed for differentiating the right and left outflow tracts (RVOT/LVOT) arrhythmia origins from 12-lead electrocardiograms (ECGs); however, the procedure is complicated. A deep learning (DL) model, a form of artificial intelligence, can directly use ECGs and depict the importance of the leads and waveforms. This study aimed to create a visualized DL model that could classify arrhythmia origins more accurately.Methods and Results: This study enrolled 80 patients who underwent catheter ablation. A convolutional neural network-based model that could classify arrhythmia origins with 12-lead ECGs and visualize the leads that contributed to the diagnosis using a gradient-weighted class activation mapping method was developed. The average prediction results of the origins by the DL model were 89.4% (88.2–90.6) for accuracy and 95.2% (94.3–96.2) for recall, which were significantly better than when a conventional algorithm is used. The ratio of the contribution to the prediction differed between RVOT and LVOT origins. Although leads V1 to V3 and the limb leads had a focused balance in the LVOT group, the contribution ratio of leads aVR, aVL, and aVF was higher in the RVOT group.Conclusions: This study diagnosed the arrhythmia origins more accurately than the conventional algorithm, and clarified which part of the 12-lead waveforms contributed to the diagnosis. The visualized DL model was convincing and may play a role in understanding the pathogenesis of arrhythmias.

Inflammation has been suggested to play a key role in the pathogenesis of atrial fibrillation (AF). Our hypothesis was that this inflammation, mediated by intermediate monocytes and toll-like receptor 4 (TLR4), causes the formation and expansion of low-voltage zones (LVZs). Prior to ablation, the monocyte subsets of 78 AF patients and TLR4 expression of 66 AF patients were analyzed via a flow cytometric analysis. Based on the CD14/CD16 expression, the monocytes were divided into three subsets: classical, intermediate, and non-classical. At the beginning of the ablation session, voltage mapping was performed. LVZs were defined as all bipolar electrogram amplitudes of

Introduction: It has been reported that ventricular tachycardia (VT) non-inducibility at the end of ablation is associated with less likely VT recurrence. However, it is not clear whether we should use VT non-inducibility as routine end point in VT ablation. The aim of this study was to evaluate VT recurrence in patients in whom VT non-inducibility could not be achieved at the end of the RF ablation and the factors attributing to the VT recurrence. METHODS and RESULTS: We analyzed 84 consecutive patients that underwent RF ablation, and 64 patients in whom VT non-inducibility could not be achieved were studied. The primary endpoint was recurrence of any sustained VT during the follow-up. During a median follow-up period of 1.4 years (IQR:0.3-2.0), 22 (34%) of the cases had VT recurrences. In the multivariate analysis showed that an LVEF≥35% (HR:0.21; 95% CI:0.07- 0.54; P

Background: Some of atrial fibrillation (AF) drivers are found in normal/mild late-gadolinium enhancement (LGE) areas, as well as moderate ones. The atrial wall thickness (AWT) has been reported to be important as a possible AF substrate. However, the AWT and degree of LGEs as an AF substrate has not been fully validated in humans. Objective: The purpose of this study was to evaluate the impact of the AWT in normal/mild LGE areas on AF drivers. Methods: A total of 287 segments in 15 persistent AF patients were assessed. AF drivers were defined as non-passively activated areas (NPAs), where rotational activation was frequently observed, and were detected by the novel real-time phase mapping (ExTRa Mapping), mild LGE areas were defined as areas with a volume ratio of the enhancement voxel of 0% to

Background A successful Radiofrequency (RF) ablation of ventricular tachycardia (VT) can prevent VT recurrence. It has been reported that VT non-inducibility at the end of RF ablation is associated with less likely VT recurrence in ischemic cardiomyopathy (ICM) and non-ICM (NCIM). However, it is not clear whether we should use VT non-inducibility as routine end point in RF ablation of VT. Purpose The aim of this study was to evaluate VT recurrence in patients who couldn't be achieved VT non-inducibility at the end of RF ablation and the factors attributed to VT recurrence in ICM and NICM patients. Methods Between January 2009 and April 2020, 84 consecutive patients (ICM: 34, NICM: 50) underwent RF ablation for drug-resistant VT in our hospital. VT non-inducibility was defined as any ventricular tachy-arrhythmia, including clinical VT, non-clinical VT, and VF, was not induced by programed stimuli at the end of session. Non-inducibility was achieved in 37 patients but it was not achieved in 47 patients (ICM: 18, NICM: 29). To evaluate the validity of “non-inducibility” as an end point of VT ablation, 47 patients (male: 40, mean age: 66±15 years) in whom non-inducibility of any ventricular tachyarrhythmia was not achieved were studied. The primary endpoint was recurrence of any sustained VT and VF during follow up period (mean follow-up period was 1.4 (range, 0.0, 2.0) years.) Results Mean left ventricular ejection fraction (LVEF) was 36±13%. Epicardial ablation was required in 8 patients. 32 patients had electrical storm at the time of ablation. Among them, 21 patients had VT recurrence and 26 patients had non-VT recurrence during follow-up period. VT recurrence rate was significantly lower in patients with LVEF≥35% than those with LVEF Conclusions Even if non-inducibility of any ventricular tachyarrhythmia wasn't achieved at the end of ablation, the patients with LVEF≥35% or who had ablated of VT isthmus might prevent VT recurrence. The validity of non-inducibility of any ventricular tachyarrhythmia should be evaluated in each patient's background. Funding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Abbott, Medtronic

A 41‐year‐old man with persistent atrial fibrillation (AF) underwent radiofrequency (RF) catheter ablation using an online real‐time phase mapping system: ExTRa Mapping. Box isolation could not terminate AF. Subsequently, RF applications on nonpassively activated areas (NPAs), where rotational activations were frequently observed, at the posterior bottom of left atrium outside of box lesion could convert AF to common atrial flutter. Of interest, the NPA near the posterior bottom were located on the patchy fibrotic tissue area assessed by the late‐gadolinium enhancement magnetic resonance imaging. This indicated the possibility of the critical AF rotor meandering through the fibrotic tissue area.

Atrial standstill (AS) is a rare condition characterized by the absence of electrical and mechanical atrial activity,1 first described by Cushny.2 The absence of P waves and bradycardia, along with a wide QRS complex, are seen on the electrocardiogram (ECG). The clinical course of AS varies greatly; the timeline can be persistent or transient, and the spatial distribution of the atrial lesion can be diffuse or partial.3, 4 In many cases, the AS appears initially in the right atrium (RA) and progresses to the entire left atrium. It occasionally complicates symptomatic sick sinus syndrome.5 We hereby describe a case of AS where a dual-chamber permanent pacemaker implantation was necessary owing to symptomatic sick sinus syndrome. An electrophysiological study (EPS) revealed inactivity throughout almost the entire RA, with the exception of the coronary sinus (CS). A permanent atrial lead was therefore placed in the CS.

Background: Some of atrial fibrillation (AF) drivers are found in lesser late-gadolinium enhancement (LGE) areas, as well as heterogenous ones. The atrial wall thickness (AWT) has been reported to be important as a possible AF substrate. However, the AWT and degree of LGEs as an AF substrate has not been fully validated in humans. Objective: The purpose of this study was to evaluate the impact of the AWT in lesser LGE areas on AF drivers. Methods: A total of 287 segments in 15 persistent AF patients were assessed. AF drivers were defined as non-passively activated areas (NPAs), where rotational activation was frequently observed, and were detected by the novel real-time phase mapping (ExTRa Mapping). Lesser LGE areas were defined as areas with a volume ratio of the enhancement voxel of

Background: A computational model demonstrated that atrial fibrillation (AF) rotors could be distributed in patchy fibrotic tissue and play an important role in AF drivers. However, this was not validated in humans. Objective: The purpose of this study was to evaluate the fibrotic tissue properties of AF rotors in patients with persistent AF. Methods: A total of 287 segments in 15 patients with persistent AF (longstanding persistent AF in 9 patients) that underwent AF ablation were assessed. Non-passively activated areas (NPAs), where rotational activation (AF rotor) was frequently observed, were detected by the novel real-time phase mapping (ExTRa Mapping). Atrial fibrosis was detected by late-gadolinium enhancement magnetic resonance imaging (LGE-MRI), and the fibrotic heterogeneity and density were assessed by the entropy (LGE-entropy) and volume ratio of the enhancement voxel (LGE-volume ratio), respectively. Results: NPAs were found in 61 (21%) of 287 segments and were mostly found around the pulmonary vein antrum. A receiver operating characteristic curve analysis yielded an optimal cutoff value of 5.7 and 10% for the LGE-entropy and LGE-volume ratio, respectively. The incidence of NPAs was significantly higher at segments with an LGE-entropy of >5.7 and LGE-volume ratio of >10% than at the other segments (38 [30%] of 126 vs. 23 [14%] of 161 segments, p = 0.001). No NPAs were found at segments with an LGE-volume ratio of >50% regardless of the LGE-entropy. Conclusion: AF rotors are mostly distributed in relatively weak and much more heterogenous fibrotic tissue.

Background: Computer simulation model demonstrated that atrial fibrillation (AF) driver attached to the patchy fibrosis assessed by late gadolinium enhancement magnetic resonance imaging (LGE-MRI). However, it has not been well elucidated in patients with persistent AF. The aim of this study is to investigate whether radiofrequency (RF) application on the patchy LGE site (PLS) could terminate AF or convert to atrial tachycardia (AT) and improve the rhythm outcome. Methods: A total of 31consecutive persistent AF patients with PLS were enrolled (PLS ablation group, mean age: 69 ± 8 years, mean left atrial diameter: 42 ± 6 mm). AF direct termination or AT conversion during RF application on the PLS were defined as favorable response. The rhythm outcome was compared between the PLS ablation group and the propensity matched conventional ablation group. Results: Favorable response was found in 15 (48%) of 31 patients (AF termination in 7, AT conversion in 8 patients). AF recurrence at 12 months follow-up was significantly less in the PLS group as compared to the control group (4 (13%) of 31 patients vs. 11 (35%) of 31 patients, log-rank p = 0.019). In patients with favorable response, AT recurred in 1 (7%) of 15 patients but AF. Conclusions: The PLS ablation could terminate AF or convert to AT in half of the patients and improve the rhythm outcome as compared to the conventional ablation. No AF recurrence was documented in patients with a favorable response.

Background Medium‐dose (25 gray) x‐ray radiation therapy has recently been performed on patients with refractory ventricular tachyarrhythmias. Unlike x‐ray, carbon ion and proton beam radiation can deliver most of their energy to the target tissues. This study investigated the electrophysiological and pathological changes caused by medium‐dose carbon ion and proton beam radiation in the left ventricle (LV). Methods and Results External beam radiation in the whole LV was performed in 32 rabbits. A total of 9 rabbits were not irradiated (control). At the 3‐month or 6‐month follow‐up, the animals underwent an open‐chest electrophysiological study and were euthanized for histological analyses. No acute death occurred. Significant LV dysfunction was not seen. The surface ECG revealed a significant reduction in the P and QRS wave voltages in the radiation groups. The electrophysiological study showed that the local conduction times in each LV site were significantly longer and that the local LV bipolar voltages were significantly lower in the radiation groups than in the control rabbits. Histologically, apoptosis, fibrotic changes, and a decrease in the expression of the connexin 43 protein were seen in the LV myocardium. These changes were obvious at 3 months, and the effects were sustained 6 months after radiation. No histological changes were seen in the coronary artery and esophagus, but partial radiation pneumonitis was observed. Conclusions Medium‐dose carbon ion and proton beam radiation in the whole LV resulted in a significant electrophysiological disturbance and pathological changes in the myocardium. Radiation of the arrhythmogenic substrate would modify the electrical status and potentially induce the antiarrhythmic effect.

Asian patients with nonvalvular atrial fibrillation (NVAF) have a higher risk of stroke and systemic embolism than non-Asians, but experience more frequent major bleeding on oral anticoagulation (OAC), including intracranial hemorrhage. Although Asians constituted only a minority of patients in

Active DNA transposons are important tools for gene functional analysis. The endogenous non-autonomous transposon, nDart1-0, in rice (Oryza sativa L.) is expected to generate various transposon-insertion mutants because nDart1-0 elements tend to insert into genic regions under natural growth conditions. We have developed a specific method (nDart1-0-iPCR) for efficient detection of nDart1-0 insertions and successfully identified the SNOW-WHITE LEAF1 (SWL1) gene in a variegated albino (swl1-v) mutant obtained from the nDart1-promoted rice tagging line. The variegated albino phenotype was caused by insertion and excision of nDart1-0 in the 5'-untranslated region of the SWL1 gene predicted to encode an unknown protein with the N-terminal chloroplast transit peptide. SWL1 expression was detected in various rice tissues at different developmental stages. However, immunoblot analysis indicated that SWL1 protein accumulation was strictly regulated in a tissue-specific manner. In the swl1 mutant, formations of grana and stroma thylakoids and prolamellar bodies were inhibited. This study revealed that SWL1 is essential for the beginning of thylakoid membrane organization during chloroplast development. Furthermore, we provide a developmental perspective on the nDart1-promoted tagging line to characterize unidentified gene functions in rice.

International audience; The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

We found that the whole plant-specific resistance (WPSR), which was expressed in the intact leaf sheaths of susceptible rice plants, was suppressed by low temperature or abscisic acid (ABA) treatment. In this study, quantitative changes in protein expression characteristic to WPSR reaction were analyzed in susceptible rice plants, ZTS, with or without ABA treatment, against the challenge of a M. oryzae isolate Hoku1. In addition, these results were compared with those of the R gene-mediated hypersensitive response (HR) in a resistant cultivar, ZTR. Proteins extracted from leaf sheaths were separated with two-dimensional polyacrylamide gel electrophoresis, and 13 proteins were changed by fungal inoculation in WPSR and R-gene-mediated resistance, but not in plants in which WPSR was suppressed by ABA. Partial amino acid sequencing of these proteins revealed that they contained - 1,3-glucanase, a thaumatin-like protein precursor (a member of the pathogenesis-related protein [PR-] group 5), and the root-specific PR-10. In transgenic ZTS plants expressing anti-sense RNA of the thaumatin-like protein, WPSR was significantly suppressed, suggesting that the induction of the thaumatin-like protein after the fungal inoculation is associated with the expression of WPSR in the susceptible rice plant.

Colletotrichum higginsianum is a hemibiotrophic fungal pathogen that causes anthracnose disease on Arabidopsis and other crucifer hosts. By exploiting natural variation in Arabidopsis we identified a resistance locus that is shared by four geographically distinct accessions (Ws-0, Kondara, Gifu-2 and Can-0). A combination of quantitative trait loci (QTL) and Mendelian mapping positioned this locus within the major recognition gene complex MRC-J on chromosome 5 containing the Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR-NB-LRR) genes RPS4 and RRS1 that confer dual resistance to C. higginsianum in Ws-0 (Narusaka et al., 2009). We find that the resistance shared by these diverse Arabidopsis accessions is expressed at an early stage of fungal invasion, at the level of appressorial penetration and establishment of intracellular biotrophic hyphae, and that this determines disease progression. Resistance is not associated with host hypersensitive cell death, an oxidative burst or callose deposition in epidermal cells but requires the defense regulator EDS1, highlighting new functions of TIR-NB-LRR genes and EDS1 in limiting early establishment of fungal biotrophy. While the Arabidopsis accession Ler-0 is fully susceptible to C. higginsianum infection, Col-0 displays intermediate resistance that also maps to MRC-J. By analysis of null mutants of RPS4 and RRS1 in Col-0 we show that these genes, individually, do not contribute strongly to C. higginsianum resistance but are both required for resistance to Pseudomonas syringae bacteria expressing the Type III effector, AvrRps4. We conclude that distinct allelic forms of RPS4 and RRS1 probably cooperate to confer resistance to different pathogens.

Generation of stage-specific cDNA libraries is a powerful approach to identify pathogen genes that are differentially expressed during plant infection. Biotrophic pathogens develop specialized infection structures inside living plant cells, but sampling the transcriptome of these structures is problematic due to the low ratio of fungal to plant RNA, and the lack of efficient methods to isolate them from infected plants. Here we established a method, based on fluorescence-activated cell sorting (FACS), to purify the intracellular biotrophic hyphae of Colletotrichum higginsianum from homogenates of infected Arabidopsis leaves. Specific selection of viable hyphae using a fluorescent vital marker provided intact RNA for cDNA library construction. Pilot-scale sequencing showed that the library was enriched with plant-induced and pathogenicity-related fungal genes, including some encoding small, soluble secreted proteins that represent candidate fungal effectors. The high purity of the hyphae (94%) prevented contamination of the library by sequences derived from host cells or other fungal cell types. RT-PCR confirmed that genes identified in the FACS-purified hyphae were also expressed in planta. The method has wide applicability for isolating the infection structures of other plant pathogens, and will facilitate cell-specific transcriptome analysis via deep sequencing and microarray hybridization, as well as proteomic analyses.

Agrobacterium tumefaciens–mediated transformation (ATMT) was used for random insertional mutagenesis to identify pathogenicity genes in the hemibiotrophic fungus Colletotrichum higginsianum. A high-throughput primary infection assay on Arabidopsis thaliana seedlings allowed the rapid screening of 8,850 transformants. Forty mutants showing reproducible pathogenicity defects on Arabidopsis and Brassica plants were obtained, and their infection phenotypes were characterized microscopically. Six mutants were impaired in appressorial melanization, fifteen had reduced penetration ability, 14 induced host papillae or hypersensitive cell death, and five were affected in the transition from biotrophy to necrotrophy. Southern blot analysis showed 58% of the transformants had single-site T-DNA integrations. Right-border flanking sequences were recovered from 12 mutants by inverse polymerase chain reaction (PCR) or thermal asymmetric interlaced PCR and were used to isolate the tagged genes from a genomic library. The putative pathogenicity genes encoded homologs of a major facilitator superfamily phosphate transporter, importin-β2, ornithine decarboxylase, β-1,3(4)-glucanase, ATP-binding endoribonuclease, carbamoyl-phosphate synthetase, and the polyprotein precursor of N-acetylglutamate kinase and N-acetylglutamyl-phosphate reductase. Six further loci were homologous to proteins of unknown function. None of these genes were previously implicated in the pathogenicity of any Colletotrichum species. The results demonstrate that ATMT is an effective tool for gene discovery in this model pathogen.

An endogenous 0.6-kb rice DNA transposon, nDart1-0, was found as an active nonautonomous element in a mutable virescent line, pyl-v, displaying leaf variegations. Here, we demonstrated that the active autonomous element aDart in pyl-v corresponds to Dart1-27 on chromosome 6 in Nipponbare, which carries no active aDart elements, and that aDart and Dart1-27 are identical in their sequences and chromosomal locations, indicating that Dart1-27 is epigenetically silenced in Nipponbare. The identification of aDart in pyl-v was first performed by map-based cloning and by detection of the accumulated transposase transcripts. Subsequently, various transposition activities of the cloned Dart1-27 element from Nipponbare were demonstrated in Arabidopsis. Dart1-27 in Arabidopsis was able to excise nDart1-0 and Dart1-27 from cloned sites, generating footprints, and to integrate into new sites, generating 8-bp target site duplications. In addition to Dart1-27, Nipponbare contains 37 putative autonomous Dart1 elements because their putative transposase genes carry no apparent nonsense or frameshift mutations. Of these, at least four elements were shown to become active aDart elements in transgenic Arabidopsis plants, even though considerable sequence divergence arose among their transposases. Thus, these four Dart1 elements and Dart1-27 in Nipponbare must be potential autonomous elements silenced epigenetically. The regulatory and evolutionary implications of the autonomous Dart1 elements and the development of an efficient transposon-tagging system in rice are discussed. Electronic supplementary material The online version of this article (doi:10.1007/s00438-008-0410-x) contains supplementary material, which is available to authorized users.

The hemibiotrophic ascomycete Colletotrichum higginsianum causes anthracnose disease on brassica crops and the model plant Arabidopsis. Melanized appressoria pierce the host cuticle and cell wall to form specialized biotrophic hyphae inside living epidermal cells. To identify proteins secreted by appressoria that may function as virulence effectors, a cDNA library was prepared from mature appressoria formed in vitro. Bidirectional sequencing of 980 clones generated 1442 high-quality expressed sequence tags (ESTs), comprising 518 unique sequences. blastx analysis showed that 353 (68 %) of these had significant similarity to entries in the NCBI non-redundant protein database, of which 49 were also homologous to experimentally verified fungal pathogenicity genes. ORFs were predicted ab initio from the unique sequences and screened for potential signal peptides using SignalP. Fifty-three unique sequences (10 %) were predicted to encode proteins entering the secretory pathway, of which 26 were likely to be soluble secreted proteins. For a selected subset of these, RT-PCR showed that seven genes that encode secreted proteins of unknown function, including two Colletotrichum-specific genes, are upregulated in appressoria and expressed early during plant infection, and therefore represent candidate effectors.

While characterized mutable alleles caused by DNA transposons have been abundant in maize since the discovery of Dissociation conferring variegation by Barbara McClintock, only a few mutable alleles have been described in rice even though the rice genome contains various transposons. Here, we show that a spontaneous mutable virescent allele, pyl-v, is caused by the disruption of the nuclear-coded essential chloroplast protease gene, OsClpP5, due to insertion of a 607-bp non-autonomous DNA transposon, non-autonomous DNA-based active rice transposon one (nDart1), belonging to the hAT superfamily. The transposition of nDart1 can be induced by crossing with a line containing an autonomous element, aDart, and stabilized by segregating out of aDart. We also identified a novel mutable dwarf allele thl-m caused by an insertion of nDart1. The japonica cultivar Nipponbare carries no aDart, although it contains epigenetically silenced Dart element(s), which can be activated by 5-azacytidine. Nipponbare bears four subgroups of about 3.6-kb Dart-like sequences, three of which contain potential transposase genes, and around 3.6-kb elements without an apparent transposase gene, as well as three subgroups of about 0.6-kb nDart1-related elements that are all internal deletions of the Dart-like sequences. Both nDart1 and 3.6-kb Dart-like elements were also present in indica varieties 93-11 and Kasalath. nDart1 appears to be the most active mutagen among nDart1-related elements contributing to generating natural variations. A candidate for an autonomous element, aDart, and a possible application of nDart1 for transposon tagging are discussed.

Mycosphaerella blight, caused by Mycosphaerella pinodes, is one of the major diseases of cultivated pea (Pisum sativum L.). To isolate the genes that are up- and down-regulated during spore germination, suppression subtraction hybridization (SSH) was performed between ungerminated and germinated spores. The 232 and 128 clones from forward and reverse libraries, respectively, were collected, sequenced, and analyzed with a BLASTX homology search. About 95% of the 32 selected clones were expressed during spore germination on a paper sheet and during infection of pea leaves. We discuss the applicability of the SSH libraries for analyzing M. pinodes genes involved in the early stage of infection.

Fusarium oxysporum f. sp. lycopersici produces extracellular enzymes known as tomatinase, which degrade the tomato phytoanticipin α-tomatine to less toxic metabolites. Tomatinase gene ( FoToml ) has been found also in certain strains of F. oxysporum belonging to formae speciales nonpathogenic on tomato plants. In the current study, four FoToml -positive strains (#2, #3, #11, and #17) of F. oxysporum belonging to formae speciales different from f. sp. lycopersici and f. sp. radicis - lycopersici were examined for their ability to infect tomato plants. All of the four strains colonized not only the roots and basal stems but also upper stems of tomato plants. One of the four strains, #3 (f. sp. batatas ), was transformed with the β-glucuronidase (GUS) reporter gene. A GUS-marked #3 (#3-GUS) colonized vascular vessels of all tomato cultivars tested differing in the extents of colonization in the stems among the cultivars. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that each FoToml of the four FoToml -positive strains was expressed in the roots and stems of tomato plants, suggesting that FoToml is important for the four strains to survive in tomato plants. To see systemic induction of defense genes in tomato plants inoculated with either #2, #3, #11, or #17, accumulations of transcripts of β-1,3-glucanase and chitinase genes were analyzed. Tomato plants inoculated with #2 or #3 showed enhanced expression of acidic chitinase gene ( Chi 3). Prior inoculation of tomato plants with either #2 or #3 suppressed the vascular wilt of tomato caused by subsequent challenge with F. oxysporum f. sp. lycopersici .