Biology subjects

Utsushi, H.

Publications and source records attributed to Utsushi, H..

9 recordsLinked to original sources

bioRxiv2025

Genomic prediction models based on a large-scale recombinant population allow quick breeding of high-yield rice

Rapid development of cultivars with optimal genome-wide allele combinations is essential for addressing agricultural challenges. However, constructing desired genotypes through conventional cross-breeding requires many generations, calling for a more efficient methodology. Here, we present a rapid breeding strategy that combines a large-scale recombinant population as starting material with interpretable genomic prediction models trained on that population. This approach enables efficient construction of target genotypes optimized for multiple traits in cultivars. To validate our strategy in rice (Oryza sativa), we established a nested association mapping population of 2,787 recombinant inbred lines derived from an elite cultivar 'Hitomebore' and 19 diverse donors. We built highly accurate genomic prediction models using this population. We then used the models to estimate haplotype-specific effects and account for trade-offs among yield-related traits, and selected optimal lines and designed breeding schemes. Crossbreeding based on these schemes produced rice lines with the target genotypes for multiple yield-related traits, supporting the predicted effects and validating the effectiveness of our strategy. This genomic breeding approach provides a general framework for rapidly breeding cultivars able to meet the challenges posed by a changing environment.

genetics↗

bioRxiv2025

Multipartite coevolution shapes plant apoplastic immunity against rice blast fungus

{beta}-1,3-glucan-binding proteins (GBPs) play crucial roles in cell wall integrity, host- pathogen interactions, and stress responses in fungi, making them essential for fungal survival and virulence. Here, we show that a GBP of the causal agent for blast fungus, Magnaporthe oryzae, is involved in multipartite coevolutionary arms race with rice (Oryza sativa). The rice thaumatin-like pathogenesis-related protein 5 (OsPR5) binds to M. oryzae GBP and sequesters it from the fungal cell wall to confer immunity, but this interaction is counteracted by thaumatin-binding proteins secreted by the fungus to suppress apoplastic immunity. Moreover, a rice receptor kinase located at the cell surface has evolved a thaumatin-like domain to detect the fungal GBP and activate immunity. Our findings reveal that GBPs and thaumatin-domain-containing proteins are engaged in intricate coevolutionary dynamics in the rice apoplast. Given the conservation of thaumatin across plants, we propose a general defense mechanism against fungal pathogens.

plant biology↗

bioRxiv2025

Fine-tuning quantitative agronomic traits by manipulating gene copy number in rice

Although recent plant pan-genome studies have revealed extensive copy number variations, there are limited studies addressing the phenotypic consequences of these variations. Here, we manipulated the copy number of OsMADS18 in rice (Oryza sativa) through gene editing and evaluated the effects. OsMADS18, which encodes a MADS-box transcription factor known to regulate plant development, is tandemly duplicated in the elite rice cultivar Hitomebore as a natural variant. We therefore edited the OsMADS18 copy number using a basic CRISPR/Cas9 system and established rice lines harboring one to three tandem copies of OsMADS18, as identified through high throughput qPCR screening. The presence of one to three OsMADS18 tandem copies was reflected in stepwise increases in transcript levels and concomitant trait values for leaf blade length and culm length. These results demonstrate that manipulating gene copy number can fine-tune important agronomic traits, providing a novel breeding strategy for crop improvement.

plant biology↗

bioRxiv2024

Impact of rice GENERAL REGULATORY FACTOR14h (GF14h) on low-temperature seed germination and its application to breeding

Direct seeding is employed to circumvent the labor-intensive process of rice (Oryza sativa) transplantation, but this approach requires varieties with vigorous low-temperature germination (LTG) when sown in cold climates. To investigate the genetic basis of LTG, we identified the quantitative trait locus (QTL) qLTG11 from rice variety Arroz da Terra, which shows rapid seed germination at lower temperatures, using QTL-seq. We delineated the candidate region to a 52-kb interval containing GENERAL REGULATORY FACTOR14h (GF14h) gene, which is expressed during seed germination. The Arroz da Terra GF14h allele encodes functional GF14h, whereas Japanese rice variety Hitomebore harbors a 4-bp deletion in the coding region. Knocking out functional GF14h in a near-isogenic line (NIL) carrying the Arroz da Terra allele decreased LTG, whereas overexpressing functional GF14h in Hitomebore increased LTG, indicating that GF14h is the causal gene behind qLTG11. Analysis of numerous Japanese rice accessions revealed that the functional GF14h allele was lost from popular varieties during modern breeding. We generated a NIL in the Hitomebore background carrying a 172-kb genomic fragment from Arroz da Terra including GF14h. The NIL showed superior LTG compared to Hitomebore, with otherwise comparable agronomic traits. The functional GF14h allele from Arroz da Terra represents a valuable resource for direct seeding in cold regions. Author SummaryRice serves as a fundamental crop sustaining over half of the global population. With the rapid growth of the worlds population, it will become increasingly important to improve rice productivity. On the other hand, the aging of rice farmers in Japan has resulted in a constant labor shortage. To address this, direct seeding, in which seeds are sown directly in rice fields without going through the most labor-intensive part of the rice cultivation process, i.e., seedling production and transplanting, has been recommended. However, prevalent elite rice varieties are known to be unsuitable for direct seeding due to their poor seed germination ability under low-temperature conditions. In this study, we show for the first time that GF14h gene from the Portuguese variety Arroz da Terra improves seed germination at low temperatures (LTG). In addition, a novel cross-bred line was generated by introducing the GF14h-containing genomic segment from Arroz da Terra into Hitomebore, a widely cultivated variety in northern Japan. This line is expected to be used as a pre-breeding material to enhance LTG. This study will provide a genetic basis for LTG and contribute to basic and applied research progress.

plant biology↗

bioRxiv2022

Apoplastic CBM1-interacting proteins bind conserved carbohydrate binding module 1 motifs in fungal hydrolases to counter pathogen invasion

When infecting plants, fungal pathogens secrete cell wall degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsCBMIP binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsCBMIP to various CBM1-containing enzymes, suggesting it has a general role in inhibiting the catalytic activities of fungal enzymes. OsCBMIP is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown of OsCBMIP reduced rice defense against M. oryzae, demonstrating that inhibition of CBM1-containing fungal enzymes by OsCBMIP is crucial for rice defense. We also identified additional CBMIP-related proteins from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism. SummaryPlants have evolved various activity-inhibiting proteins as a defense against fungal cell wall degrading enzymes (CWDEs), but how plants counteract the function of fungal enzymes containing carbohydrate binding modules (CBMs) remains unknown. Here, we demonstrate that OsCBMIP, a member of the cysteine-rich repeat secretion protein family, interacts with fungal CBM1. OsCBMIP binding to CBM1 of a blast fungal xylanase blocks access to cellulose, resulting in the inhibition of xylanase enzymatic activity. Our study provides significant insights into plant countermeasure against CWDEs in the apoplastic space during plant-fungal pathogen interactions. It also reveals a molecular function of the DUF26 domain widely distributed in plant proteins.

plant biology↗

bioRxiv2021

A Jacalin-like lectin-domain-containing protein of Sclerospora graminicola act as an apoplastic virulence effector in plant-oomycete interactions

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant- pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the Jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from S. graminicola (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs via protein mass spectrometry analysis of extracellular fluid from S. graminicola-inoculated foxtail millet leaves. SgJRLs consist of a Jacalin-like lectin domain and an N-terminal putative secretion signal, and SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with GFP localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defense-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defense responses.

plant biology↗

bioRxiv2020

Lumi-Map, a real-time luciferase bioluminescence screen of mutants combined with MutMap, reveals Arabidopsis genes involved in PAMP-triggered immunity

Plants recognize pathogen-associated molecular patterns (PAMPs) to activate PAMP-triggered immunity (PTI). However, our knowledge of PTI signaling remains limited. In this report, we introduce Lumi-Map, a high-throughput platform for identifying causative single nucleotide polymorphisms (SNPs) to studying PTI signaling components. In Lumi-Map, a transgenic reporter plant line is produced that contains a firefly luciferase (LUC) gene driven by a defense gene promoter, which generates luminescence upon PAMP treatment. The line is mutagenized and the mutants with altered luminescence patterns are screened by a high-throughput real-time bioluminescence monitoring system. Selected mutants are subjected to MutMap analysis, a whole genome sequencing (WGS)-based method of rapid mutation identification, to identify the causative SNP responsible for the luminescence pattern change. We generated nine transgenic Arabidopsis reporter lines expressing LUC gene fused to multiple promoter sequences of defense-related genes. These lines generate luminescence upon activation of FLAGELLIN-SENSING 2 (FLS2) by flg22, a PAMP derived from bacterial flagellin. We selected the WRKY29-promoter reporter line to identify mutants in the signaling pathway downstream of FLS2. After screening 24,000 ethylmethanesulfonate (EMS)-induced mutants of the reporter line, we isolated 22 mutants with altered WRKY29 expression upon flg22 treatment (abbreviated as awf mutants). While five flg22-insensitive awf mutants harbored mutations in FLS2 itself, Lumi-Map revealed three genes not previously associated with PTI. Lumi-Map has the potential to identify novel PAMPs and their receptors as well as signaling components downstream of the receptors.

plant biology↗

bioRxiv2017

An unconventional NOI/RIN4 domain of a rice NLR protein binds host EXO70 protein to confer fungal immunity

As much as 10% of plant immune receptors from the nucleotide-binding domain leucine-rich repeat (NLR) family carry integrated domains (IDs) that can directly bind pathogen effectors. However, it remains unclear whether direct binding to effectors is a universal feature of ID-containing NLRs given that only a few NLR-IDs have been functionally characterized. Here we show that the rice (Oryza sativa) sensor NLR-ID Pii2 confers resistance to strains of the rice blast fungus Magnaporthe oryzae that carry the effector AVR-Pii without directly binding this protein. First, we show that AVR-Pii binds the exocyst subunit OsExo70F2 in rice (Oryza sativa) to dissociate preformed complexes of OsExo70F2 with host RPM1 INTERACTING PROTEIN4 (RIN4) at the conserved NOI motif, facilitating a possible virulence function. Second, we show that in its resting state, Pii2 binds OsExo70F2 and OsExo70F3, essential components of Pii-mediated resistance, through its integrated NOI domain. Remarkably, AVR-Pii binding to OsExo70F2/F3 leads to dissociation of the Pii2-OsExo70F2 and Pii2-OsExo70F3 complexes, destabilization of Pii2, and activation of immunity. These findings support a novel conceptual model in which an NLR-ID monitors alterations of tethered host proteins targeted by pathogen effectors, providing insight into pathogen recognition mechanisms. Significance statementPlant diseases diminish crop yields by over 20% each year, and deploying resistant crops is the most effective way to combat them. Nucleotide-binding domain leucine-rich repeat (NLR)-type receptors are the major player in plant resistance against pathogens, with a subset of NLRs containing unconventional domains called integrated domains (ID) derived from host proteins. Previous studies suggest that pathogen avirulence (AVR) effectors directly bind or modify NLR-IDs before they are recognized by the host. Here, we reveal that the rice NLR-ID receptor Pii2 indirectly recognizes AVR-Pii when the effector dissociates Pii2 from the host Exo70 proteins tethered to Pii2. We propose a new model of how NLRs can recognize pathogens, expanding our understanding of plant immunity.

plant biology↗

bioRxiv2017

Rice blast resistance gene Pii is controlled by a pair of NBS-LRR genes Pii-1 and Pii-2

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are conserved cytosolic receptors that recognize pathogen effectors and trigger immunity in plants. Recent studies indicate that NLRs function in pairs. Rice resistance gene Pii has been known to confer resistance against rice blast pathogen Magnaporthe oryzae carrying AVR-Pii. Previously we reported isolation of Pii gene from the rice cultivar Hitomebore (Takagi et al. 2013). To further understand rice components required for Pii-mediated resistance, we screened 5,600 mutant lines of Hitomebore cultivar and identified two mutants that lost Pii resistance without any changes in Pii gene sequence. Application of MutMap-Gap, the whole genome sequencing-based method of mutation identification, to the two mutants revealed that they have mutations in another NLR gene located close to Pii. The F1 plants derived from a cross of the two mutants showed pii phenotype, demonstrating that the newly identified NLR gene is indeed a component of Pii resistance. We thus designate the previously isolated Pii gene as Pii-1 and the newly isolated NLR gene as Pii-2.

genetics↗