RESUMO
The advanced model of floral morphogenesis is based largely on data from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa), but this process is less well understood in the Triticeae. Here, we investigated a sterile barley (Hordeum vulgare) mutant with malformed floral organs (designated mfo1), of which the paleae, lodicules, and stamens in each floret were all converted into lemma-like organs, and the ovary was abnormally shaped. Combining bulked-segregant analysis, whole-genome resequencing, and TILLING approaches, the mfo1 mutant was attributed to loss-of-function mutations in the MADS-box transcription factor gene HvAGL6, a key regulator in the ABCDE floral morphogenesis model. Through transcriptomic analysis between young inflorescences of wild-type and mfo1 plants, 380 genes were identified as differentially expressed, most of which function in DNA binding, protein dimerization, cell differentiation, or meristem determinacy. Regulatory pathway enrichment showed HvAGL6 associates with transcriptional abundance of many MADS-box genes, including the B-class gene HvMADS4. Mutants with deficiency in HvMADS4 exhibited the conversion of stamens into supernumerary pistils, producing multiple ovaries resembling the completely sterile multiple ovaries 3.h (mov3.h) mutant. These findings demonstrate that the regulatory model of floral morphogenesis is conserved across plant species and provides insights into the interactions between HvAGL6 and other MADS-box regulators.
RESUMO
Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen-plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.
Assuntos
Glucose , Xanthomonas campestris , Virulência , Glucose/metabolismo , Plantas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Sacarose/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologiaRESUMO
The soil-borne yellow mosaic virus disease, which is caused by the bymoviruses barley yellow mosaic virus (BaYMV) and/or barley mild mosaic virus (BaMMV), seriously threatens winter barley production in Europe and East Asia. Both viruses are transmitted by the soil-borne plasmodiophorid Polymyxa graminis and are difficult to eliminate through chemical or physical measures in the field, making breeding for resistant cultivars the optimal strategy for disease control. The resistance locus rym1/11 was cloned encoding the host factor gene Protein Disulfide Isomerase Like 5-1 (PDIL5-1), whose loss-of-function variants confer broad-spectrum resistance to multiple strains of BaMMV/BaYMV. Most resistance-conferring variants have been identified in six-rowed barley landraces/historic cultivars, and their introgression into modern two-rowed malting cultivars is difficult because PDIL5-1 is located in a peri-centromeric region with suppressed recombination. In this study, we used CRISPR/Cas9 genome editing to modify PDIL5-1 in the BaYMV/BaMMV-susceptible elite malting barley cv. 'Golden Promise' and obtained the mutants pdil5-1-a and pdil5-1-b. PDIL5-1 in the pdil5-1-a mutant encodes a protein lacking a cysteine residue, and pdil5-1-b contains a protein-coding frameshift. Both mutants were completely resistant to BaYMV. The knockout mutant pdil5-1-b showed complete BaMMV resistance, while pdil5-1-a showed decreased viral accumulation but no disease symptoms if compared to 'Golden Promise'. Both PDIL5-1 edited lines, as well as the previously produced EMS-induced pdil5-1 mutant '10253-1-5' in the elite malting barley cv. 'Barke' background, displayed no growth or yield penalties in garden experiments or bymovirus-free field trials. Line '10253-1-5' showed improved resistance and yield performance compared to the wild-type and its sibling line when grown in infectious fields. Therefore, genome editing of the host factor gene PDIL5-1 could facilitate the breeding of barley varieties with resistance to bymoviruses.
RESUMO
Barley is a diploid species with a genome smaller than those of other members of the Triticeae tribe, making it an attractive model for genetic studies in Triticeae crops. The recent development of barley genomics has created a need for a high-throughput platform to identify genetically uniform mutants for gene function investigations. In this study, we report an ethyl methanesulfonate (EMS)-mutagenized population consisting of 8525 M3 lines in the barley landrace "Hatiexi" (HTX), which we complement with a high-quality de novo assembly of a reference genome for this genotype. The mutation rate within the population ranged from 1.51 to 4.09 mutations per megabase, depending on the treatment dosage of EMS and the mutation discrimination platform used for genotype analysis. We implemented a three-dimensional DNA pooling strategy combined with multiplexed amplicon sequencing to create a highly efficient and cost-effective TILLING (targeting induced locus lesion in genomes) platform in barley. Mutations were successfully identified from 72 mixed amplicons within a DNA pool containing 64 individual mutants and from 56 mixed amplicons within a pool containing 144 individuals. We discovered abundant allelic mutants for dozens of genes, including the barley Green Revolution contributor gene Brassinosteroid insensitive 1 (BRI1). As a proof of concept, we rapidly determined the causal gene responsible for a chlorotic mutant by following the MutMap strategy, demonstrating the value of this resource to support forward and reverse genetic studies in barley.
Assuntos
Hordeum , Metanossulfonato de Etila/farmacologia , Hordeum/genética , Mutagênese , Mutação , Genética ReversaRESUMO
The WRKY transcription factors (WRKYs) are known for their crucial roles in biotic and abiotic stress responses, and developmental and physiological processes. In barley, early studies revealed their importance, whereas their diversity at the population scale remains hardly estimated. In this study, 98 HsWRKYs and 103 HvWRKYs have been identified from the reference genome of wild and cultivated barley, respectively. The tandem duplication and segmental duplication events from the cultivated barley were observed. By taking advantage of early released exome-captured sequencing datasets in 90 wild barley accessions and 137 landraces, the diversity analysis uncovered synonymous and non-synonymous variants instead of loss-of-function mutations that had occurred at all WRKYs. For majority of WRKYs, the haplotype and nucleotide diversity both decreased in cultivated barley relative to the wild population. Five WRKYs were detected to have undergone selection, among which haplotypes of WRKY9 were enriched, correlating with the geographic collection sites. Collectively, profiting from the state-of-the-art barley genomic resources, this work represented the characterization and diversity of barley WRKY transcription factors, shedding light on future deciphering of their roles in barley domestication and adaptation.
Assuntos
Hordeum/genética , Fatores de Transcrição/genética , Aclimatação , Domesticação , Duplicação Gênica , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genoma de Planta , Genômica , Haplótipos , Filogenia , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismoRESUMO
Plant-specific TEOSINTE BRANCHED 1/CYCLOIDEA/PROLIFERATING CELL FACTORS 1/2 (TCP) transcription factors have known roles in inflorescence architecture. In barley, there are two family members INTERMEDIUM-C (INT-c/HvTB1-1) and COMPOSITUM 1 (COM1/HvTCP24) which are involved in the manipulation of spike architecture, whereas the participation of TCP family genes in selection from wild (Hordeum vulgare subsp. spontaneum, Hs) to cultivated barley (Hordeum vulgare subsp. vulgare, Hv) remains poorly investigated. Here, by conducting a genome-wide survey for TCP-like sequences in publicly-released datasets, 22 HsTCP and 20 HvTCP genes encoded for mature proteins were identified and assigned into two classes (I and II) based on their functional domains and the phylogenetic analysis. Each counterpart of the orthologous gene in wild and cultivated barley usually represented a similarity on the transcriptional profile across the tissues. The diversity analysis of TCPs in 90 wild barley accessions and 137 landraces with geographically-referenced passport information revealed the detectable selection at three loci including INT-c/HvTB1-1, HvPCF2, and HvPCF8. Especially, the HvPCF8 haplotypes in cultivated barley were found correlating with their geographical collection sites. There was no difference observed in either transactivation activity in yeast or subcellular localization in Nicotiana benthamiana among these haplotypes. Nevertheless, the genome-wide diversity analysis of barley TCP genes in wild and cultivated populations provided insight for future functional characterization in plant development such as spike architecture.
Assuntos
Hordeum/genética , Proteínas de Plantas/genética , Polimorfismo Genético , Seleção Artificial , Fatores de Transcrição/genética , Flores/genética , Flores/crescimento & desenvolvimento , Haplótipos , Hordeum/crescimento & desenvolvimento , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Seleção Genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Bymovirus-induced yellow mosaic diseases seriously threaten global production of autumn-sown barley and wheat, which are two of the presently most important crops around the world. Under natural field conditions, the diseases are caused by infection of soil-borne plasmodiophorid Polymyxa graminis-transmitted bymoviruses of the genus Bymovirus of the family Potyviridae. Focusing on barley and wheat, this article summarizes the achievements on taxonomy, geography and host specificity of these disease-conferring viruses, as well as the genetics of resistance in barley, wheat and wild relatives. Moreover, based on recent progress of barley and wheat genomics, germplasm resources and large-scale sequencing, the exploration and isolation of corresponding resistant genes from wheat and barley as well as relatives, no matter what a large and complicated genome is present, are becoming feasible and are discussed. Furthermore, the foreseen advances on cloning of the resistance or susceptibility-encoding genes, which will provide the possibility to explore the functional interaction between host plants and soil-borne viral pathogens, are discussed as well as the benefits for marker-assisted resistance breeding in barley and wheat.
Assuntos
Resistência à Doença/imunologia , Regulação da Expressão Gênica de Plantas , Hordeum/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Potyviridae/isolamento & purificação , Triticum/imunologia , Resistência à Doença/genética , Genoma Viral , Hordeum/genética , Hordeum/virologia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Triticum/genética , Triticum/virologiaRESUMO
During plant-pathogen interactions, pathogenic bacteria have evolved multiple strategies to cope with the sophisticated defence systems of host plants. Proline iminopeptidase (PIP) is essential to Xanthomonas campestris pv. campestris (Xcc) virulence, and is conserved in many plant-associated bacteria, but its pathogenic mechanism remains unclear. In this study, we found that disruption of pip in Xcc enhanced its flagella-mediated bacterial motility by decreasing intracellular bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) levels, whereas overexpression of pip in Xcc restricted its bacterial motility by elevating c-di-GMP levels. We also found that PIP is a type III secretion system-dependent effector capable of eliciting a hypersensitive response in non-host, but not host plants. When we transformed pip into the host plant Arabidopsis, higher bacterial titres were observed in pip-overexpressing plants relative to wild-type plants after Xcc inoculation. The repressive function of PIP on plant immunity was dependent on PIP's enzymatic activity and acted through interference with the salicylic acid (SA) biosynthetic and regulatory genes. Thus, PIP simultaneously regulates two distinct regulatory networks during plant-microbe interactions, i.e. it affects intracellular c-di-GMP levels to coordinate bacterial behaviour, such as motility, and functions as a type III effector translocated into plant cells to suppress plant immunity. Both processes provide bacteria with the regulatory potential to rapidly adapt to complex environments, to utilize limited resources for growth and survival in a cost-efficient manner and to improve the chances of bacterial survival by helping pathogens to inhabit the internal tissues of host plants.
RESUMO
Quorum sensing (QS) signals are used by bacteria to regulate biological functions in response to cell population densities. Cyclic diguanosine monophosphate (c-di-GMP) regulates cell functions in response to diverse environmental chemical and physical signals that bacteria perceive. In Burkholderia cenocepacia, the QS signal receptor RpfR degrades intracellular c-di-GMP when it senses the QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible signal factor (BDSF), as a proxy for high cell density. However, it was unclear how this resulted in control of BDSF-regulated phenotypes. Here, we found that RpfR forms a complex with a regulator named GtrR (BCAL1536) to enhance its binding to target gene promoters under circumstances where the BDSF signal binds to RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and inhibits its ability to control gene expression. Mutations in rpfR and gtrR had overlapping effects on both the B. cenocepacia transcriptome and BDSF-regulated phenotypes, including motility, biofilm formation, and virulence. These results show that RpfR is a QS signal receptor that also functions as a c-di-GMP sensor. This protein thus allows B. cenocepacia to integrate information about its physical and chemical surroundings as well as its population density to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and gamma proteobacteria.
Assuntos
Proteínas de Bactérias/genética , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/patogenicidade , GMP Cíclico/análogos & derivados , Ácidos Graxos Monoinsaturados/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de Quorum/genética , Animais , Carga Bacteriana , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Infecções por Burkholderia/microbiologia , Infecções por Burkholderia/patologia , Burkholderia cenocepacia/crescimento & desenvolvimento , GMP Cíclico/metabolismo , Camundongos , Mutação , Fenótipo , Transdução de Sinais , VirulênciaRESUMO
The widespread communications between prokaryotes and eukaryotes via signaling molecules are believed to affect gene expression in both partners. During the communication process, the contacted organisms produce and release small molecules that establish communication channels between two kingdoms-this procedure is known as interkingdom signaling. Interkingdom communications are widespread between pathogenic or beneficial bacteria and their host plants, with diversified outcomes depending on the specific chemical-triggered signaling pathways. Deciphering the signals or language of this interkingdom communication and uncovering the underlying mechanisms are major current challenges in this field. It is evident that diverse signaling molecules can be produced or derived from bacteria and plants, and researchers have sought to identify these signals and explore the mechanisms of the signaling pathways. The results of such studies will lead to the development of strategies to improve plant disease resistance through controlling interkingdom signals, rather than directly killing the pathogenic bacteria. Also, the identification of signals produced by beneficial bacteria will be useful for agricultural applications. In this review, we summarize the recent progress of cross-kingdom interactions between plant and bacteria, and how LuxR-family transcription factors in plant associated bacterial quorum sensing system are involved in the interkingdom signaling.
Assuntos
Fenômenos Fisiológicos Bacterianos , Doenças das Plantas/prevenção & controle , Plantas/microbiologia , Resistência à Doença , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , Percepção de Quorum , Transdução de SinaisRESUMO
Xanthomonas campestris pv. campestris strain 8004 contains an orphan quorum-sensing (QS) locus, xccR-pip(Xcc), in which the proline iminopeptidase (pip(Xcc)) gene (where "Xcc" indicates that the pip gene is from X. campestris pv. campestris) is positively regulated by the LuxR homologue XccR by binding to the luxXc box of the pip(Xcc) promoter. The disruption of pip(Xcc) significantly attenuated the virulence of X. campestris pv. campestris. An imperfect plant-inducible promoter (PIP) box is located in the upstream region of the pip(Xcc) promoter, which is the putative binding site of the transcriptional activator HrpX. To explore whether the expression of the pip(Xcc) gene is regulated by HrpX, the expression level of a pip(Xcc) promoter-gusA fusion gene was assayed in an hrpX disruption mutant. The results showed that the lack of HrpX dramatically decreased the ß-glucuronidase (GUS) activity. Further analyses using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR indicated that the imperfect PIP box in X. campestris pv. campestris is specifically bound to HrpX. These data demonstrated that the pip(Xcc) gene belongs to the hrp regulon and that the imperfect PIP box of the pip(Xcc) promoter could be a cis element for the HrpX protein. We further showed in a pulldown assay that XccR can bind HrpX, suggesting that these two regulatory proteins coactivate the virulence factor by binding to the different cis elements of the pip(Xcc) gene and adapt to the host environment during X. campestris pv. campestris infection.