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Summary Plant RNA virus‐based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter‐driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus‐induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)‐based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9‐mediated targeted mutagenesis in wheat and maize. BSMV‐based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV‐mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.

Phytophthora sojae is an important model species for oomycete functional genomics research. Recently, a CRISPR/Cas9‐mediated genome‐editing technology has been successfully established in P. sojae, which has been rapidly and widely applied in oomycete research. However, there is an emerging consensus in the biological community that a complete functional gene research system is needed such as developed in the investigations in functional complementation carried out in this study. We report the development of an in situ complementation method for accurate restoration of the mutated gene. We targeted a regulatory B‐subunit of protein phosphatase 2A (PsPP2Ab1) to verify this knockout and subsequent complementation system. We found that the deletion of PsPP2Ab1 in P. sojae leads to severe defects in vegetative hyphal growth, soybean infection, and loss of the ability to produce sporangia. Subsequently, the reintroduction of PsPP2Ab1 into the knockout mutant remedied all of the deficiencies. This study demonstrates the successful implementation of an in situ complementation system by CRISPR/Cas9, which will greatly accelerate functional genomics research of oomycetes in the post‐genomic era. A regulatory B‐subunit of protein phosphatase 2A was used to demonstrate an in situ complementation method of a mutated gene in Phytophthora sojae, verifying its roles in hyphal growth, soybean infection, and sporangia production.

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen‐derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens. Plants undergoing cell death during pathogen infection or in response to an immunogenic pattern emit red light that can be detected and quantified with an imaging system available in many laboratories.

Summary Transcription Activator‐Like effectors (TALes) represent the largest family of type III effectors among pathogenic bacteria and play a critical role in the process of infection. Strains of Xanthomonas oryzae pv. oryzae (Xoo) and some strains of other Xanthomonas pathogens contain large numbers of TALe genes. Previous techniques to clone individual or a complement of TALe genes through conventional strategies are inefficient and time‐consuming due to multiple genes (up to 29 copies) in a given genome, and technically challenging due to the repetitive sequences (up to 33 nearly identical 102‐nucleotide repeats) of individual TALe genes. Thus, only a limited number of TALe genes have been molecularly cloned and characterized, and the functions of most TALe genes remain unknown. Here, we present an easy and efficient cloning technique to clone TALe genes selectively through in vitro homologous recombination and single‐strand annealing, and demonstrate the feasibility of this approach with four different Xoo strains. Based on the Gibson assembly strategy, two complementary vectors with scaffolds that can preferentially capture all TALe genes from a pool of genomic fragments were designed. Both vector systems enabled cloning of a full complement of TALe genes from each of four Xoo strains and functional analysis of individual TALes in rice in approximately 1 month compared to 3 months by previously used methods. The results demonstrate a robust tool to advance TALe biology and a potential for broad usage of this approach to clone multiple copies of highly competitive DNA elements in any genome of interest.

Summary RNA silencing is a universal mechanism involved in development, epigenetic modifications and responses to biotic and abiotic stresses. The major components of this mechanism are Dicer‐like (DCL), Argonaute (AGO) and RNA‐dependent RNA polymerase (RDR) proteins. Understanding the role of each component is of great scientific and agronomic importance. Plants, including Nicotiana benthamiana, an important plant model, usually possess four DCL proteins, each of which has a specific role, namely being responsible for the production of an exclusive small RNA population. Here, we used RNA interference (RNAi) technology to target DCL proteins and produced single and combinatorial mutants for DCL. We analysed the phenotype for each DCL knockdown plant, together with the small RNA profile, by next‐generation sequencing (NGS). We also investigated transgene expression, as well as viral infections, and were able to show that DCL suppression results in distinct developmental defects, changes in small RNA populations, increases in transgene expression and, finally, higher susceptibility in certain RNA viruses. Therefore, these plants are excellent tools for the following: (i) to study the role of DCL enzymes; (ii) to overexpress proteins of interest; and (iii) to understand the complex relationship between the plant silencing mechanism and biotic or abiotic stresses.

Summary Genome sequencing of pathogenic fungi has revealed the presence of various effectors that aid pathogen invasion by the manipulation of plant immunity. Effectors are often individually dispensable because of duplication and functional redundancy as a result of the arms race between host plants and pathogens. To study effectors that have functional redundancy, multiple gene disruption is often required. However, the number of selection markers that can be used for gene targeting is limited. Here, we established a marker recycling system that allows the use of the same selection marker in successive transformations in the model fungal pathogen Colletotrichum orbiculare, a causal agent of anthracnose disease in plants belonging to the Cucurbitaceae. We identified two C. orbiculare homologues of yeast URA3/pyrG, designated as URA3A and URA3B, which can be used as selection markers on medium with no uridine. The gene can then be removed from the genome via homologous recombination when the fungus is grown in the presence of 5‐fluoroorotic acid (5‐FOA), a chemical that is converted into a toxin by URA3 activity. The ura3a/b double mutants showed auxotrophy for uridine and insensitivity to 5‐FOA. Using the ura3a/b mutants, transformation with the URA3B marker and its removal were successfully applied to disrupt the virulence‐related gene, PKS1. The pks1 mutants showed a reduction in virulence, demonstrating that the method can be used to study virulence‐related genes in C. orbiculare. The establishment of a URA3‐based marker recycling system in plant‐pathogenic fungi enables the genetic analysis of multiple genes that have redundant functions, including effector genes.

Background Since the leucocyte-platelet rich fibrin (L-PRF) was published in 2001, many studies have been developed, analyzing its properties, and also verifying new possibilities to improve it. Thereby, it emerges the advanced-platelet rich fibrin (A-PRF) with a protocol that optimizes the properties obtained by the L-PRF. Nonetheless, there is a gap in the literature to landmark the evolutive process concerning the mechanical properties in specific the resistance to tensile strength which consequently may influence the time for membrane degradation. Thus, this study had the goal to compare the resistance to the traction of membranes produced with the original L-PRF and A-PRF protocols, being the first to this direct comparison. Findings The harvest of blood from a healthy single person, with no history of anticoagulant usage. We performed the protocols described in the literature, within a total of 13 membranes produced for each protocol ( n = 26). Afterward, the membranes were prepared and submitted to a traction test assessing the maximal and the average traction achieved for each membrane. The data were analyzed statistically using the unpaired t test. Regarding average traction, A-PRF obtained a value of 0.0288 N mm −2 and L-PRF 0.0192 N mm −2 ( p < 0.05 using unpaired t test). For maximal traction, A-PRF obtained 0.0752 N mm −2 and L-PRF 0.0425 N mm −2 ( p < 0.05 using unpaired t test). Conclusion With this study, it was possible to conclude that indeed A-PRF has a significative higher maximal traction score and higher average traction compared to L-PRF, indicating that it had a higher resistance when two opposing forces are applied.

Background The aim of this study was to introduce a complete 3D workflow for immediate implant retained prosthetic rehabilitation following maxillectomy in cancer surgery. The workflow consists of a 3D virtual surgical planning for tumor resection, zygomatic implant placement, and for an implant-retained prosthetic-obturator to fit the planned outcome situation for immediate loading. Materials and methods In this study, 3D virtual surgical planning and resection of the maxilla, followed by guided placement of 10 zygomatic implants, using custom cutting and drill/placement-guides, was performed on 5 fresh frozen human cadavers. A preoperatively digitally designed and printed obturator prosthesis was placed and connected to the zygomatic implants. The accuracy of the implant positioning was obtained using 3D deviation analysis by merging the pre- and post-operative CT scan datasets. Results The preoperatively designed and manufactured obturator prostheses matched accurately the per-operative implant positions. All five obturators could be placed and fixated for immediate loading. The mean prosthetic point deviation on the cadavers was 1.03 ± 0.85 mm; the mean entry point deviation was 1.20 ± 0.62 mm; and the 3D angle deviation was 2.97 ± 1.44°. Conclusions It is possible to 3D plan and accurately execute the ablative surgery, placement of zygomatic implants, and immediate placement of an implant-retained obturator prosthesis with 3D virtual surgical planning.The next step is to apply the workflow in the operating room in patients planned for maxillectomy.

Purpose Dental implants may become displaced into the maxillary sinus due to insufficient primary stability, changes in nasal air pressure, or surrounding bone resorption and should be removed as soon as possible. The aim of this study was to evaluate the efficacy of the modified endoscopic sinus surgery (MESS) approach for removal of displaced dental implants. Methods From September 2010 to November 2021, we studied 15 cases with displaced implants in the maxillary sinus. The patient characteristics, medical history, clinical and imaging results, and post-removal outcomes were retrospectively assessed. Results The symptoms included sinusitis (100%), pain (26.6%), postnasal drip (6.6%), nasal obstruction (26.6%), and oroantral communication (26.6%). Two cases were managed through the crestal approach (13.3%), while two cases were treated with the Caldwell–Luc procedure (13.3%). One case was addressed using functional endoscopic sinus surgery (6.7%), while 10 cases were managed with the MESS approach (66.7%). MESS allows functional rehabilitation of mucociliary clearance by the cilia in the sinus membrane. Implant displacement into the maxillary sinus can be classified as early, late, or delayed displacement. Conclusions MESS is a reliable treatment option that can identify migrated dental implants in any part of the sinus with endoscopic assistance for functional rehabilitation of the maxillary sinus without postoperative sequelae.

Introduction There are few zygomatic implants (ZI) designs available. The objective of this non-interventional study was to report the effectiveness of two new site-specific ZI, selected and placed following the zygoma anatomy-guided approach (ZAGA). Materials and methods Consecutive patients presenting indications for rehabilitation using ZI were treated according to ZAGA Concept recommendations. Implants were immediately loaded following the manufacturer’s instructions. Success criteria regarding prosthetic offset, rhino-sinus status, soft tissue condition, and implant stability were additionally used as outcome parameters. Results Twenty patients were followed for a period of 12 to 28 months (average 18.8 months). Ten received 2 ZI plus regular anterior implants; One received 3 ZI plus regular implants and nine received 4 ZI. In total, 59 ZI were placed, 34 (58%) Straumann ZAGA-Flat design, and 25 (42%) ZAGA-Round. Forty-nine percent of the sites were classified as ZAGA-4 type and 27% as ZAGA-2. Four patients (20%) presented discontinuities of the sinus–nose floor before surgery and 15 patients (75%) presented previous sinus opacities. All implants bar one reached more than 45 N.cm of insertion torque. No surgical complications were observed. After 1 year, the modified Lund–Mackay score was negative in 17 patients. Seventeen sites in 11 patients exhibited decreased opacity when pre-surgical imaging was compared to 1-year post-surgical CBCT. All implants and prostheses remained stable and in function. Conclusions The study concluded 100% implant/prosthesis survival rates and low complication levels. Within the limitations of the sample and observation period, results suggest that even in cases of extremely resorbed maxillae (as per cases in this study), ZAGA-Flat and ZAGA-Round ZI are viable treatment options when restoring atrophic maxillae following the ZAGA protocol.