The Hessian fly resistance gene HvRHF1 is localized in an NBS-LRR gene cluster in barley.

Hessian fly (Mayetiola destructor Say) is a significant pest in cereal crops, causing substantial yield losses worldwide. While host resistance is the most efficient method for pest control, research on genetic characterization of Hessian fly resistance in barley (Hordeum vulgare L.) has been limited, and the underlying resistance mechanism remains largely unknown. In this study, we conducted fine mapping of a crucial Hessian fly resistance locus, known as HvRHF1, using a biparental population. Assisted with genetic markers and robust phenotyping assay, we pinpointed the HvRHF1 gene to an ~ 82 kb region on chromosome 4H. Gene prediction and annotation revealed that the HvRHF1 locus comprises three complete NBS-LRR genes, which are characteristic of disease resistance genes. As a result, our study not only provides valuable resources for resistance in barley and genetic tools for breeding, but also identifies candidate genes that lay the foundation for cloning HvRHF1. This endeavor will significantly contribute to our understanding of the molecular mechanisms underlying cereal resistance to Hessian fly.

Plastid terminal oxidase is required for chloroplast biogenesis in barley.

Chloroplast biogenesis is critical for crop biomass and economic yield. However, chloroplast development is a very complicated process coordinated by cross-communication between the nucleus and plastids, and the underlying mechanisms have not been fully revealed. To explore the regulatory machinery for chloroplast biogenesis, we conducted map-based cloning of the Grandpa 1 (Gpa1) gene regulating chloroplast development in barley. The spontaneous mutation gpa1.a caused a variegation phenotype of the leaf, dwarfed growth, reduced grain yield, and increased tiller number. Genetic mapping anchored the Gpa1 gene onto 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. One gene (HORVU.MOREX.r3.2HG0213170) in the delimited region encodes a putative plastid terminal oxidase (PTOX) in thylakoid membranes, which is homologous to IMMUTANS (IM) of Arabidopsis. The IM gene is required for chloroplast biogenesis and maintenance of functional thylakoids in Arabidopsis. Using CRISPR technology and gene transformation, we functionally validated that the PTOX-encoding gene, HORVU.MOREX.r3.2HG0213170, is the causal gene of Gpa1. Gene expression and chemical analysis revealed that the carotenoid biosynthesis pathway is suppressed by the gpa1 mutation, rendering mutants vulnerable to photobleaching. Our results showed that the overtillering associated with the gpa1 mutation was caused by the lower accumulation of carotenoid-derived strigolactones (SLs) in the mutant. The cloning of Gpa1 not only improves our understanding of the molecular mechanisms underlying chloroplast biosynthesis but also indicates that the PTOX activity is conserved between monocots and dicots for the establishment of the photosynthesis factory.

Genome-Wide Association Study of Host Resistance to Hessian Fly in Barley.

The Hessian fly (HF), Mayetiola destructor (Diptera: Cecidomyiidae), is one of the most devastating insect pests of cereals including wheat, barley, and rye. Although wheat is the preferred host for HF, this continuously evolving pest has been emerging as a threat to barley production. However, characterization and identification of genetic resistance to HF has not been conducted in barley. In the present study, we used a genome-wide association study (GWAS) to identify barley resistance loci to HF using a geographically diverse set of 234 barley accessions. The results showed that around 90% of barley lines were highly susceptible, indicating a significant vulnerability to HF in barley, and a total of 29 accessions were resistant, serving as potential resistance resources. GWAS with a mixed linear model revealed two marker-trait associations, both on chromosome 4H. The resistance loci and associated markers will facilitate barley improvement and development for breeders. In addition, our results are fundamental for genetic studies to understand the HF resistance mechanism in barley.

Targeting Disease Susceptibility Genes in Wheat Through wide Hybridization with Maize Expressing Cas9 and Guide RNA.

Two genes (TaHRC and Tsn1) conferring susceptibility to Fusarium head blight and tan spot, Septoria nodorum blotch, and spot blotch in wheat were targeted through wide hybridization with maize expressing Cas9 and guide RNA (gRNA). For each gene, two target sites were selected and corresponding gRNA expression cassettes were synthesized and cloned into a binary vector carrying the CRISPR/Cas9-mediated genome editing machinery. The constructed binary vectors were used to transform the hybrid maize Hi-II through an Agrobacterium-mediated approach to generate T0 and T1 plants, which were used to cross with wheat variety Dayn for targeting Tsn1 or the susceptible allele (TaHRC-S) of TaHRC as well as with the near-isogenic line (Day-Fhb1) of Dayn for targeting the resistant allele (TaHRC-R) of TaHRC. Haploid embryos were rescued in vitro from the wide crosses to generate haploid plants. PCR amplification and sequencing indicated that 15 to 33% of the haploid plants contained the target gene with mutations at the target sites. This wheat × maize hybridization combined with genome editing approach provides a useful alternative tool, not only for targeting susceptibility genes to improve disease resistance without regulatory issues, but also for understanding gene function in wheat. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genetic and physical localization of a major susceptibility gene to Pyrenophora teres f. maculata in barley.

KEY MESSAGE: Genetic characterization of a major spot form net blotch susceptibility locus to using linkage mapping to identify a candidate gene and user-friendly markers in barley. Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is an economically important foliar diseases in barley. Although various resistance loci have been identified, breeding for SFNB-resistant varieties has been hampered due to the complex virulence profile of Ptm populations. One resistance locus in the host may be effective against one specific isolate, but it may confer susceptibility to other isolates. A major susceptibility QTL on chromosome 7H, named Sptm1, was consistently identified in many studies. In the present study, we conduct fine mapping to localize Sptm1 with high resolution. A segregating population was developed from selected F2 progenies of the cross Tradition (S) × PI 67381 (R), in which the disease phenotype was determined by the Sptm1 locus alone. Disease phenotypes of critical recombinants were confirmed in the following two consecutive generations. Genetic mapping anchored the Sptm1 gene to an ⁓400 kb region on chromosome 7H. Gene prediction and annotation identified six protein-coding genes in the delimited Sptm1 region, and the gene encoding a putative cold-responsive protein kinase was selected as a strong candidate. Therefore, providing fine localization and candidate of Sptm1 for functional validation, our study will facilitate the understanding of susceptibility mechanism underlying the barley-Ptm interaction and offers a potential target for gene editing to develop valuable materials with broad-spectrum resistance to SFNB.

Paired Medicago receptors mediate broad-spectrum resistance to nodulation by Sinorhizobium meliloti carrying a species-specific gene.

Plants have evolved the ability to distinguish between symbiotic and pathogenic microbial signals. However, potentially cooperative plant-microbe interactions often abort due to incompatible signaling. The Nodulation Specificity 1 (NS1) locus in the legume Medicago truncatula blocks tissue invasion and root nodule induction by many strains of the nitrogen-fixing symbiont Sinorhizobium meliloti. Controlling this strain-specific nodulation blockade are two genes at the NS1 locus, designated NS1a and NS1b, which encode malectin-like leucine-rich repeat receptor kinases. Expression of NS1a and NS1b is induced upon inoculation by both compatible and incompatible Sinorhizobium strains and is dependent on host perception of bacterial nodulation (Nod) factors. Both presence/absence and sequence polymorphisms of the paired receptors contribute to the evolution and functional diversification of the NS1 locus. A bacterial gene, designated rns1, is required for activation of NS1-mediated nodulation restriction. rns1 encodes a type I-secreted protein and is present in approximately 50% of the nearly 250 sequenced S. meliloti strains but not found in over 60 sequenced strains from the closely related species Sinorhizobium medicae. S. meliloti strains lacking functional rns1 are able to evade NS1-mediated nodulation blockade.

Host and pathogen genetics reveal an inverse gene-for-gene association in the P. teres f. maculata-barley pathosystem.

KEY MESSAGE: Pathogen and host genetics were used to uncover an inverse gene-for-gene interaction where virulence genes from the pathogen Pyrenophora teres f. maculata target barley susceptibility genes, resulting in disease. Although models have been proposed to broadly explain how plants and pathogens interact and coevolve, each interaction evolves independently, resulting in various scenarios of host manipulation and plant defense. Spot form net blotch is a foliar disease of barley caused by Pyrenophora teres f. maculata. We developed a barley population (Hockett × PI 67381) segregating for resistance to a diverse set of P. teres f. maculata isolates. Quantitative trait locus analysis identified major loci on barley chromosomes (Chr) 2H and 7H associated with resistance/susceptibility. Subsequently, we used avirulent and virulent P. teres f. maculata isolates to develop a pathogen population, identifying two major virulence loci located on Chr1 and Chr2. To further characterize this host-pathogen interaction, progeny from the pathogen population harboring virulence alleles at either the Chr1 or Chr2 locus was phenotyped on the Hockett × PI 67381 population. Progeny harboring only the Chr1 virulence allele lost the barley Chr7H association but maintained the 2H association. Conversely, isolates harboring only the Chr2 virulence allele lost the barley Chr2H association but maintained the 7H association. Hockett × PI 67381 F2 individuals showed susceptible/resistant ratios not significantly different than 15:1 and results from F2 inoculations using the single virulence genotypes were not significantly different from a 3:1 (S:R) ratio, indicating two dominant susceptibility genes. Collectively, this work shows that P. teres f. maculata virulence alleles at the Chr1 and Chr2 loci are targeting the barley 2H and 7H susceptibility alleles in an inverse gene-for-gene manner to facilitate colonization.

Electrochemical Noise Measurement to Assess Corrosion of Steel Reinforcement in Concrete.

The electrochemical noise method (ENM) has previously been employed to monitor the corrosion of steel reinforcement in concrete. The development of solid-state Ag/AgCl-based probes and dedicated monitoring technology (ProCoMeter) now offers a wider range of ENM configurations. The present study involves the laboratory investigation of three mortar samples containing steel bars and varying additions of chloride, with a view to future field application. ENM could be used to provide corrosion information on reinforcement without the need to provide direct electrical connections to the steel and without the risk or inducing or increasing corrosion. In addition to half-cell potentials, measurements were made using ENM in three different probe configurations over a total test period of 90 days. The samples were then broken open and the bars extracted and cleaned. A comparison was then made between the calculated metal thickness loss obtained from the Rn values and the actual metal thickness loss. The results showed that each configuration was able to order the results in the expected manner, with the simple single substrate (SSS) arrangement providing the best correlation with direct measurements. The study is ongoing with the intention of measurements being obtained in situ on existing reinforced concrete structures.

Genetic mapping of host resistance to the Pyrenophora teres f. maculata isolate 13IM8.3.

Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is a foliar disease of barley that results in significant yield losses in major growing regions worldwide. Understanding the host-parasite interactions between pathogen virulence/avirulence genes and the corresponding host susceptibility/resistance genes is important for the deployment of genetic resistance against SFNB. Two recombinant inbred mapping populations were developed to characterize genetic resistance/susceptibility to the Ptm isolate 13IM8.3, which was collected from Idaho (ID). An Illumina Infinium array was used to produce a genome-wide marker set. Quantitative trait loci (QTL) analysis identified ten significant resistance/susceptibility loci, with two of the QTL being common to both populations. One of the QTL on 5H appears to be novel, while the remaining loci have been reported previously. Single nucleotide polymorphisms (SNPs) closely linked to or delimiting the significant QTL have been converted to user-friendly markers. Loci and associated molecular markers identified in this study will be useful in genetic mapping and deployment of the genetic resistance to SFNB in barley.

Genetic analysis of the barley variegation mutant, grandpa1.a.

BACKGROUND: Providing the photosynthesis factory for plants, chloroplasts are critical for crop biomass and economic yield. However, chloroplast development is a complicated process, coordinated by the cross-communication between the nucleus and plastids, and the underlying biogenesis mechanism has not been fully revealed. Variegation mutants have provided ideal models to identify genes or factors involved in chloroplast development. Well-developed chloroplasts are present in the green tissue areas, while the white areas contain undifferentiated plastids that are deficient in chlorophyll. Unlike albino plants, variegation mutants survive to maturity and enable investigation into the signaling pathways underlying chloroplast biogenesis. The allelic variegated mutants in barley, grandpa 1 (gpa1), have long been identified but have not been genetically characterized. RESULTS: We characterized and genetically analyzed the grandpa1.a (gpa1.a) mutant. The chloroplast ultrastructure was evaluated using transmission electron microscopy (TEM), and it was confirmed that chloroplast biogenesis was disrupted in the white sections of gpa1.a. To determine the precise position of Gpa1, a high-resolution genetic map was constructed. Segregating individuals were genotyped with the barley 50 k iSelect SNP Array, and the linked SNPs were converted to PCR-based markers for genetic mapping. The Gpa1 gene was mapped to chromosome 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. In the variegated gpa1.a mutant, we identified a large deletion in this gene cluster that eliminates a putative plastid terminal oxidase (PTOX). CONCLUSIONS: Here we characterized and genetically mapped the gpa1.a mutation causing a variegation phenotype in barley. The PTOX-encoding gene in the delimited region is a promising candidate for Gpa1. Therefore, the present study provides a foundation for the cloning of Gpa1, which will elevate our understanding of the molecular mechanisms underlying chloroplast biogenesis, particularly in monocot plants.

Genome Sequence of Streptomyces sp. Strain HB-N217, Isolated from the Marine Sponge Forcepia sp.

The genome sequence of the Forcepia sponge-derived bacterium Streptomyces sp. strain HB-N217 was determined, with approximately 8.25 Mbp and a G+C content of 72.1%. Thirty biosynthetic gene clusters that bear the capability to produce secondary metabolites were predicted. The results will aid marine natural product chemistry and sponge-microbe association studies.

Genetic and Physical Localization of the Gene Controlling Leaf Pigmentation Pattern in Medicago truncatula.

In Medicago truncatula, some ecotypes form a black or purple stain in the middle of adaxial leaf surface due to accumulation of anthocyanins. However, this morphological marker is missing in some other ecotypes, although anthocyanin biosynthesis pathway is not disrupted. Genetic analysis indicated that the lack of the leaf spot of anthocyanins accumulation is a dominant trait, which is controlled by a single gene, LPP1 Genetic mapping indicated that the LPP1 gene was delimited to a 280 kb-region on Chromosome 7. A total of 8 protein-coding genes were identified in the LPP1 locus through gene annotation and sequence analysis. Of those, two genes, putatively encoding MYB-transcriptional suppressors, were selected as candidates for functional validation.

Genetic localization of the SPC gene controlling pod coiling direction in Medicago truncatula.

BACKGROUND: Handedness in plants introduced by helical growth of organs is frequently observed, and it has fascinated plant scientists for decades. However, the genetic control of natural handedness has not been revealed. In the model legume Medicago truncatula, pods can be coiled in a clockwise or anti-clockwise manner, providing a model for genetic analysis of plant handedness. OBJECTIVE: We aimed to localize the Sense of Pod Coiling (SPC) gene controlling pod coiling direction in M. truncatula. METHODS: Linkage analysis was used with a biparental population for fine mapping of the SPC gene. The genome sequence of M. truncatula Mt4.0 was used for marker identification and physical mapping. Single nucleotide polymorphisms (SNPs) between the parental lines were converted to CAPS (cleaved amplified polymorphic sequences) markers. Genetic map was constructed using the software JoinMap version 3.0. Gene predication and annotation provided by the M. truncatula genome database (http://www.medicagogenome.org) was confirmed with the programs of FGENESH and Pfam 32.0, respectively. Quantitative reverse transcription PCR (qRT-PCR) was used to analyze the relative expression levels of candidate genes. RESULTS: The genetic analysis indicated that the anti-clockwise coiling is dominant to clockwise and is controlled by the single gene, SPC. The SPC gene was delimited to a 250 kb-region on Chromosome 7. Total of 15 protein-coding genes were identified in the SPC locus through gene annotation and sequence analysis. Of those, two genes, potentially encoding a receptor-like kinase and a vacuolar cation/proton exchanger respectively, were selected as candidates for the SPC gene. CONCLUSIONS: The result presented here lay a foundation for gene cloning of SPC, which will help us to understand the molecular mechanisms underlying helical growth in plant organs.

Antagonistic regulation of axillary bud outgrowth by the BRANCHED genes in tobacco.

As a key integrator of shoot branching, BRANCHED 1 (BRC1) coordinates and is orchestrated by endogenous and environmental signals involved in the regulation of axillary bud outgrowth. In the present study, we characterized the regulatory roles of five BRC gene members in tobacco (Nicotiana tabacum L.) using CRISPR site-directed mutagenesis and overexpression assays. It was shown that lateral branching was negatively regulated by NtBRC1A-1, 1B-1, and 1B-2, but was unexpectedly promoted by NtBRC2A. Suppression of bud growth may be attained by direct binding of NtBRCs to the Tassels Replace Upper Ears 1 (TRU1) genes. It was speculated that NtBRC2A probably confers a dominant negative effect by interfering with the branching-inhibitory BRC1 genes. Our results suggested that highly homologous gene family members may function antagonistically in the same signaling pathway. However, the molecular mechanism underlying NtBRC2A-mediated outgrowth of axillary buds needs to be further addressed. KEY MESSAGE: Axillary bud outgrowth in general is negatively regulated by the BRANCHED gene. Here we show that the BRANCHED genes play opposing regulatory roles in tobacco lateral branching.

Nodule-Specific Cysteine-Rich Peptides Negatively Regulate Nitrogen-Fixing Symbiosis in a Strain-Specific Manner in Medicago truncatula.

Medicago truncatula shows a high level of specificity when interacting with its symbiotic partner Sinorhizobium meliloti. This specificity is mainly manifested at the nitrogen-fixing stage of nodule development, such that a particular bacterial strain forms nitrogen-fixing nodules (Nod+/Fix+) on one plant genotype but ineffective nodules (Nod+/Fix-) on another. Recent studies have just begun to reveal the underlying molecular mechanisms that control this specificity. The S. meliloti strain A145 induces the formation of Fix+ nodules on the accession DZA315.16 but Fix- nodules on Jemalong A17. A previous study reported that the formation of Fix- nodules on Jemalong A17 by S. meliloti A145 was conditioned by a single recessive allele named Mtsym6. Here we demonstrate that the specificity associated with S. meliloti A145 is controlled by multiple genes in M. truncatula, including NFS1 and NFS2 that encode nodule-specific cysteine-rich (NCR) peptides. The two NCR peptides acted dominantly to block rather than promote nitrogen fixation by S. meliloti A145. These two NCR peptides are the same ones that negatively regulate nitrogen-fixing symbiosis associated with S. meliloti Rm41.

The Soybean Rfg1 Gene Restricts Nodulation by Sinorhizobium fredii USDA193.

Sinorhizobium fredii is a fast-growing rhizobial species that can establish a nitrogen-fixing symbiosis with a wide range of legume species including soybeans (Glycine max). In soybeans, this interaction shows a high level of specificity such that particular S. fredii strains nodulate only a limited set of plant genotypes. Here we report the identification of a dominant gene in soybeans that restricts nodulation with S. fredii USDA193. Genetic mapping in an F2 population revealed co-segregation of the underlying locus with the previously cloned Rfg1 gene. The Rfg1 allele encodes a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat class of plant resistance proteins that restricts nodulation by S. fredii strains USDA257 and USDA205, and an allelic variant of this gene also restricts nodulation by Bradyrhizobium japonicum USDA122. By means of complementation tests and CRISPR/Cas9-mediated gene knockouts, we demonstrate that the Rfg1 allele also is responsible for resistance to nodulation by S. fredii USDA193. Therefore, the Rfg1 allele likely provides broad-spectrum resistance to nodulation by many S. fredii and B. japonicum strains in soybeans.

Microsymbiont discrimination mediated by a host-secreted peptide in Medicago truncatula.

The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.

Host-secreted antimicrobial peptide enforces symbiotic selectivity in Medicago truncatula.

Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.