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Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. Plant diseases reduce harvests of the world's most important food crops including wheat, rice, potato, and corn. These diseases are important for both global food security and local subsistence farming. To fight these diseases, crops (like all plants) have an immune system that can detect the telltale molecules produced by disease-causing microbes (also known as pathogens) and mount a defence response to protect the plant. Nucleotide-binding, leucine-rich repeat receptors (or NLRs for short) are plant proteins that survey the inside of plant cells looking for these telltale molecules. These receptors have played a central role in efforts to breed disease resistance into crop plants for decades, but little is known about how they work. Maqbool, Saitoh et al. have now used a range of biochemical, structural biology and activity-based assays to study how one NLR from rice directly interacts with a molecule from the rice blast fungus. This fungus causes the most important disease of rice (called rice blast), and the fungal molecule in question is also known as an ‘effector’ protein. A technique called X-ray crystallography was used to reveal the three-dimensional structure of the effector bound to part of the NLR called the ‘integrated HMA domain’. Biochemical techniques were then used to measure how strongly the effector (and other related effectors) interacted with this domain of the NLR. These results, combined with a close examination of the three-dimensional structure, allowed a set of changes to be made to the effector that stopped it interacting with the NLR protein domain in the laboratory. Maqbool, Saitoh et al. then performed experiments in rice plants and showed that changes to the effector that stopped it interacting with the NLR domain also stopped the effector from triggering a defence response in plants. Similar results were also obtained in experiments that used the model plant Nicotiana benthamiana. In the middle of the 20th century, an American plant pathologist called Harold Henry Flor proposed that the outcomes of interactions between plants and disease-causing microbes were based on interactions between specific biological molecules. The findings of Maqbool, Saitoh et al. provide a new structural basis for this model. A detailed picture of these molecular interactions will allow researchers to engineer tailored NLRs that detect a wider range of pathogen molecules. In the future such an approach could contribute to efforts to protect the world's most important crops from plant diseases.

Although ophthalmic viscosurgical devices are quite important for safe cataract surgery, currently, postoperative residual ophthalmic viscosurgical devices can cause various complications. Previously, we developed a method to visualize residual ophthalmic viscosurgical devices after irrigation/aspiration in vitro and found that the amount of residual ophthalmic viscosurgical device on a single-piece intraocular lens was greater than that on a three-piece intraocular lens. In the present study, we compared the amounts of residual ophthalmic viscosurgical device among various foldable intraocular lenses to investigate the factors that determine the quantity of residual ophthalmic viscosurgical device. Simulated cataract surgery was performed in pig eyes using an ophthalmic viscosurgical device labeled with fluorescent silica particles. After the simulated surgery procedure, the fluorescent silica attached to the intraocular lens was observed and quantified by inductively coupled plasma-atomic emission spectrometry after intraocular lens removal. The amount of residual ophthalmic viscosurgical device was compared among five representative single-piece intraocular lenses and one three-piece intraocular lens. The distribution and amount of the residual ophthalmic viscosurgical device differed for each intraocular lens. The amount of silicon in the lens capsule differed among the intraocular lens types. The postoperative residual tendency of ophthalmic viscosurgical devices differed among various single-piece intraocular lenses. The behavior of the intraocular lenses within the capsule affected the residual tendency. The removal of ophthalmic viscosurgical device in the lens capsule should be tailored for each intraocular lens to improve efficiency.

To examine the variability in glaucoma screening using fundus images among physicians, including non-ophthalmologists. Sixty-nine eyes from 69 patients, including 25 eyes with glaucoma, were included from the Jikei University Hospital from July 2019 to December 2022. Fundus images were captured using TRC-NW8 (Topcon Corporation, Tokyo, Japan), and were interpreted by 10 non-ophthalmologists, 10 non-specialist ophthalmologists, and 9 specialists for diagnostic accuracy. We analyzed differences in diagnostic accuracy among the three groups. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Kappa coefficient were compared, using the Kruskal-Wallis test followed by a post hoc Dunn's test. The sensitivity and specificity were 0.22 and 0.92 for non-ophthalmologists, 0.49 and 0.83 for non-specialist ophthalmologists, and 0.68 and 0.87 for specialists, respectively. Both specialists and non-specialist ophthalmologists showed significantly higher sensitivity than non-ophthalmologists (Dunn's test, P<0.001 and P=0.031). There was no significant difference in specificity among the three groups (Kruskal-Wallis test, P=0.086). The PPV did not differ significantly between the groups (Kruskal-Wallis test, P=0.108), while the NPV was significantly higher in specialists compared to non-ophthalmologists (Dunn's test, P<0.001). Specialists also had a significantly higher Kappa coefficient than non-ophthalmologists and non-specialist ophthalmologists (Dunn's test, P<0.001 and P=0.024). Diagnostic accuracy varied significantly based on the physician's background.

Chiharu Wada-Koike,1,* Ryo Terauchi,1,* Kota Fukai,2 Kei Sano,1 Euido Nishijima,1 Koji Komatsu,1 Kyoko Ito,3 Tomohiro Kato,3 Masayuki Tatemichi,2 Yoshiaki Kabata,4 Tadashi Nakano11Department of Ophthalmology, The Jikei University School of Medicine, Tokyo, Japan; 2Department of Preventive Medicine, Tokai University School of Medicine, Isehara, Japan; 3Centre for Preventive Medicine, The Jikei University School of Medicine, Tokyo, Japan; 4Department of Ophthalmology, Jikei University School of Medicine, Daisan Hospital, Tokyo, Japan*These authors contributed equally to this workCorrespondence: Yoshiaki Kabata, Department of Ophthalmology, Jikei University School of Medicine, Daisan Hospital, 4-11-1 Izumihontyou, Komae-si, Tokyo, 125-8506, Japan, Tel +81 3 3480 1151, Fax +81 3 3480 5700, Email [email protected]View the original paper by Dr Wada-Koike and colleaguesThis is in response to the Letter to the Editor

We evaluated the late-onset ocular hypotensive effect of ripasudil after long-term administration in real-world clinical data and investigated its associated factors in primary open-angle glaucoma (POAG). We reviewed the clinical patients with POAG who newly started ripasudil without changes of treatment. Enrolled eyes were assigned to two groups: positive group with the late-onset effect and negative group. Eyes that show the late-onset effect 6 months after starting ripasudil were defined as positive. Logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for the late-onset effect. We enrolled 74 eyes of 74 patients with POAG (age, 67.5 ± 10.9 years; mean deviation, -11.2 ± 5.9 dB) and followed them for 14.2 ± 5.0 months. Among them, 12 (16.2%) eyes were assigned to the positive group. Retinal nerve fiber layer (RNFL) thickness (73.4 ± 12.9 vs 64.0 ± 9.8 μm, = 0.04) and primary IOP (18.8 ± 4.1 vs 15.8 ± 4.3 mmHg, = 0.01) before starting ripasudil were higher in the positive group than in the negative group. The late-onset effect was associated with higher IOP (OR, 1.22; 1.01-1.48) and thicker RNFL (2.76; 1.15-6.63). Some patients with POAG showed the late-onset IOP-lowering effect of ripasudil, and its associated factors were higher IOP and thicker RNFL. The addition of ripasudil may offer potential benefits particularly for early-stage glaucoma with thicker RNFL.

Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer—fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

The random amplified polymorphic DNA (RAPD) technique was used to determine the sex of a dioecious species, Carica papayaL., with three sex types, male, female and hermaphrodite. A 450 bp marker fragment, named PSDM(Papaya Sex Determination Marker), exists in all male and hermaphrodite plants but not in the female plants so far analyzed. The DNA sequence of PSDM exhibited no significant similarity to previously reported sequences. A sequence-characterized amplified region (SCAR) marker, SCARps, was developed from PSDM to determine the sex of papaya. Southern hybridization, using PSDM as a probe, showed that PSDM exists in the male and hermaphrodite genomes, but not in the female genome. This result strongly suggests that PSDM is located on the chromosome region that is specific to the male and the hermaphrodite. SCARps is a suitable marker for the precise and rapid diagnosis of sex in papaya.

Plants use pattern recognition receptors to defend themselves from microbial pathogens. These receptors recognize pathogen-associated molecular patterns (PAMPs) and activate signaling pathways that lead to immunity. In rice (Oryza sativa), the chitin elicitor binding protein (CEBiP) recognizes chitin oligosaccharides released from the cell walls of fungal pathogens. Here, we show that the rice blast fungus Magnaporthe oryzae overcomes this first line of plant defense by secreting an effector protein, Secreted LysM Protein1 (Slp1), during invasion of new rice cells. We demonstrate that Slp1 accumulates at the interface between the fungal cell wall and the rice plasma membrane, can bind to chitin, and is able to suppress chitin-induced plant immune responses, including generation of reactive oxygen species and plant defense gene expression. Furthermore, we show that Slp1 competes with CEBiP for binding of chitin oligosaccharides. Slp1 is required by M. oryzae for full virulence and exerts a significant effect on tissue invasion and disease lesion expansion. By contrast, gene silencing of CEBiP in rice allows M. oryzae to cause rice blast disease in the absence of Slp1. We propose that Slp1 sequesters chitin oligosaccharides to prevent PAMP-triggered immunity in rice, thereby facilitating rapid spread of the fungus within host tissue.

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.