Subfunctionalization of NRC3 altered the genetic structure of the Nicotiana NRC network.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play crucial roles in immunity against pathogens in both animals and plants. In solanaceous plants, activation of several sensor NLRs triggers their helper NLRs, known as NLR-required for cell death (NRC), to form resistosome complexes to initiate immune responses. While the sensor NLRs and downstream NRC helpers display diverse genetic compatibility, molecular evolutionary events leading to the complex network architecture remained elusive. Here, we showed that solanaceous NRC3 variants underwent subfunctionalization after the divergence of Solanum and Nicotiana, altering the genetic architecture of the NRC network in Nicotiana. Natural solanaceous NRC3 variants form three allelic groups displaying distinct compatibilities with the sensor NLR Rpi-blb2. Ancestral sequence reconstruction and analyses of natural and chimeric variants identified six key amino acids involved in sensor-helper compatibility. These residues are positioned on multiple surfaces of the resting NRC3 homodimer, collectively contributing to their compatibility with Rpi-blb2. Upon activation, Rpi-blb2-compatible NRC3 variants form membrane-associated punctate and high molecular weight complexes, and confer resistance to the late blight pathogen Phytophthora infestans. Our findings revealed how mutations in NRC alleles lead to subfunctionalization, altering sensor-helper compatibility and contributing to the increased complexity of the NRC network.

Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages.

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

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.

Phylogenomic analysis in Latilactobacillus sakei by using polymorphisms detected by next-generation sequencing.

Latilactobacillus sakei is a lactic acid bacterium used to produce a wide range of fermented food products. To understand their characteristics and adaptability to various nutrient sources, we applied strain-specific, nucleotide-concatenated (SSC) sequences to the phylogenetic analysis of 32 L. sakei strains isolated from various locations and products. SSC sequences were developed by concatenating the polymorphisms detected by whole-genome sequencing. This enabled us to use sufficient polymorphisms and avoid the bias caused by selecting partial sequences, such as that in core genome and multi-locus sequence typing. SSC sequence-based analysis revealed that the phylogenetic relations for L. sakei are based on the different nutrition sources rather than geographical distance.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins.

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 OsRMC 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 OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

Genome Analysis Revives a Forgotten Hybrid Crop Edo-dokoro in the Genus Dioscorea.

A rhizomatous Dioscorea crop 'Edo-dokoro' was described in old records of Japan, but its botanical identity has not been characterized. We found that Edo-dokoro is still produced by four farmers in Tohoku-machi of the Aomori prefecture, Japan. The rhizomes of Edo-dokoro are a delicacy to the local people and are sold in the markets. Morphological characters of Edo-dokoro suggest its hybrid origin between the two species, Dioscorea tokoro and Dioscorea tenuipes. Genome analysis revealed that Edo-dokoro likely originated by hybridization of a male D. tokoro to a female D. tenuipes, followed by a backcross with a male plant of D. tokoro. Edo-dokoro is a typical minor crop possibly maintained for more than 300 years but now almost forgotten by the public. We hypothesize that there are many such uncharacterized genetic heritages passed over generations by small-scale farmers that await serious scientific investigation for future use and improvement by using modern genomics information.

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution.

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

High-performance pipeline for MutMap and QTL-seq.

Summary: Bulked segregant analysis implemented in MutMap and QTL-seq is a powerful and efficient method to identify loci contributing to important phenotypic traits. However, the previous pipelines were not user-friendly to install and run. Here, we describe new pipelines for MutMap and QTL-seq. These updated pipelines are approximately 5-8 times faster than the previous pipeline, are easier for novice users to use, and can be easily installed through bioconda with all dependencies. Availability: The new pipelines of MutMap and QTL-seq are written in Python and can be installed via bioconda. The source code and manuals are available online (MutMap: https://github.com/YuSugihara/MutMap, QTL-seq: https://github.com/YuSugihara/QTL-seq).

Sat-BSA: an NGS-based method using local de novo assembly of long reads for rapid identification of genomic structural variations associated with agronomic traits.

Advances in next generation sequencing (NGS)-based methodologies have accelerated the identifications of simple genetic variants such as point mutations and small insertions/deletions (InDels). Structural variants (SVs) including large InDels and rearrangements provide vital sources of genetic diversity for plant breeding. However, their analysis remains a challenge due to their complex nature. Consequently, novel NGS-based approaches are needed to rapidly and accurately identify SVs. Here, we present an NGS-based bulked-segregant analysis (BSA) technique called Sat-BSA (SVs associated with traits) for identifying SVs controlling traits of interest in crops. Sat-BSA targets allele frequencies at all SNP positions to first identify candidate genomic regions associated with a trait, which is then reconstructed by long reads-based local de novo assembly. Finally, the association between SVs, RNA-seq-based gene expression patterns and trait is evaluated for multiple cultivars to narrow down the candidate genes. We applied Sat-BSA to segregating F2 progeny obtained from crosses between turnip cultivars with different tuber colors and successfully isolated two genes harboring SVs that are responsible for tuber phenotypes. The current study demonstrates the utility of Sat-BSA for the identification of SVs associated with traits of interest in species with large and heterozygous genomes.

Genome analyses reveal the hybrid origin of the staple crop white Guinea yam (Dioscorea rotundata).

White Guinea yam (Dioscorea rotundata) is an important staple tuber crop in West Africa. However, its origin remains unclear. In this study, we resequenced 336 accessions of white Guinea yam and compared them with the sequences of wild Dioscorea species using an improved reference genome sequence of D. rotundata In contrast to a previous study suggesting that D. rotundata originated from a subgroup of Dioscorea praehensilis, our results suggest a hybrid origin of white Guinea yam from crosses between the wild rainforest species D. praehensilis and the savannah-adapted species Dioscorea abyssinica We identified a greater genomic contribution from D. abyssinica in the sex chromosome of Guinea yam and extensive introgression around the SWEETIE gene. Our findings point to a complex domestication scenario for Guinea yam and highlight the importance of wild species as gene donors for improving this crop through molecular breeding.