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There has been limited research on external browning (EB) of walnut. This work discovered 1888 metabolites and 34 anthocyanins in walnut pellicles (WPs) after three drying methods using widely-targeted and anthocyanin-targeted metabolomics. Based on OPLS-DA and correlation analysis, 64 temperature-responsive metabolites (TRMs; 13 anthocyanins and 51 flavonoids) were identified as critical components in relation to EB. Notably, 14 flavonoids exhibited a strong positive correlation (r > 0.9) with the browning index (BI), with upregulation of >60% after browning. Most of the identified anthocyanins were negatively linked with BI because of degradation (>45%), with correlation coefficients ranging from 0.75 to 0.97. Furthermore, anthocyanidin reductase and laccase were the two key enzymes involved in the EB of WPs, with their activities increasing by 10.57-fold and 1.32-fold, respectively, with increasing drying temperature. A metabolic pathway network of the TRM was built to provide insights into the potential mechanisms underlying EB in WPs.
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Effector proteins secreted by bacteria that infect mammalian and plant cells often subdue eukaryotic host cell defenses by simultaneously affecting multiple targets. However, instances when a bacterial effector injected in the competing bacteria sabotage more than a single target have not been reported. Here, we demonstrate that the effector protein, LtaE, translocated by the type IV secretion system from the soil bacterium Lysobacter enzymogenes into the competing bacterium, Pseudomonas protegens, affects several targets, thus disabling the antibacterial defenses of the competitor. One LtaE target is the transcription factor, LuxR1, that regulates biosynthesis of the antimicrobial compound, orfamide A. Another target is the sigma factor, PvdS, required for biosynthesis of another antimicrobial compound, pyoverdine. Deletion of the genes involved in orfamide A and pyoverdine biosynthesis disabled the antibacterial activity of P. protegens, whereas expression of LtaE in P. protegens resulted in the near-complete loss of the antibacterial activity against L. enzymogenes. Mechanistically, LtaE inhibits the assembly of the RNA polymerase complexes with each of these proteins. The ability of LtaE to bind to LuxR1 and PvdS homologs from several Pseudomonas species suggests that it can sabotage defenses of various competitors present in the soil or on plant matter. Our study thus reveals that the multi-target effectors have evolved to subdue cell defenses not only in eukaryotic hosts but also in bacterial competitors.
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Proteínas Bacterianas , Lysobacter , Pseudomonas , Sistemas de Secreción Tipo IV , Pseudomonas/genética , Pseudomonas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Lysobacter/genética , Lysobacter/metabolismo , Sistemas de Secreción Tipo IV/genética , Sistemas de Secreción Tipo IV/metabolismo , Regulación Bacteriana de la Expresión Génica , Oligopéptidos/metabolismo , Oligopéptidos/genética , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factor sigma/genética , Factor sigma/metabolismoRESUMEN
Phytophthora pathogens possess hundreds of effector genes that exhibit diverse expression patterns during infection, yet how the expression of effector genes is precisely regulated remains largely elusive. Previous studies have identified a few potential conserved transcription factor binding sites (TFBSs) in the promoters of Phytophthora effector genes. Here, we report a MYB-related protein, PsMyb37, in Phytophthora sojae, the major causal agent of root and stem rot in soybean. Yeast one-hybrid and electrophoretic mobility shift assays showed that PsMyb37 binds to the TACATGTA motif, the most prevalent TFBS in effector gene promoters. The knockout mutant of PsMyb37 exhibited significantly reduced virulence on soybean and was more sensitive to oxidative stress. Consistently, transcriptome analysis showed that numerous effector genes associated with suppressing plant immunity or scavenging reactive oxygen species were down-regulated in the PsMyb37 knockout mutant during infection compared to the wild-type P. sojae. Several promoters of effector genes were confirmed to drive the expression of luciferase in a reporter assay. These results demonstrate that a MYB-related transcription factor contributes to the expression of effector genes in P. sojae.
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Phytophthora , Enfermedades de las Plantas , Regiones Promotoras Genéticas , Factores de Transcripción , Phytophthora/patogenicidad , Phytophthora/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Regiones Promotoras Genéticas/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/genética , Glycine max/microbiología , Glycine max/genética , Virulencia/genéticaRESUMEN
There is a scarcity of data on how the lipid composition of oily seeds changes in response to biotic stress. Yellow peach moth (Conogethes punctiferalis) has caused massive economic losses on the pecan (Carya illinoinensis) industry. Lipidomics is used in this study to determine the lipid composition of pecan and how it changes in response to insect attack. Pecan had 167 lipids, including 34 glycerolipids (GL), 62 glycerophospholipids (GP), 17 fatty acyls (FA), 41 sphingolipids (SP), and 13 saccharolipids (SL). The effects of biotic stress on lipids, particularly GL and GP, were significant. Biotic stress significantly reduced the lipid content of chains longer than 48. Forty-four significantly different lipids were discovered as potential biomarkers for distinguishing non-infected pecans from infested pecans. In addition, we used bioinformatics to identify the five most important metabolic pathways in order to investigate the processes underlying the changes. Our discoveries may offer valuable insights for enhancing pecan production in the future and contribute novel perspectives towards enhancing the nutritional value of pecans.
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Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.
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Ascomicetos , Magnaporthe , Oryza , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Oryza/metabolismo , Fenazinas/metabolismo , Enfermedades de las Plantas/genéticaRESUMEN
Oomycete pathogens secrete numerous crinkling and necrosis proteins (CRNs) to manipulate plant immunity and promote infection. However, the functional mechanism of CRN effectors is still poorly understood. Previous research has shown that the Phytophthora sojae effector PsCRN108 binds to the promoter of HSP90s and inhibits their expression, resulting in impaired plant immunity. In this study, we found that in addition to HSP90, PsCRN108 also suppressed other Heat Shock Protein (HSP) family genes, including HSP40. Interestingly, PsCRN108 inhibited the expression of NbHSP40 through its promoter, but did not directly bind to its promoter. Instead, PsCRN108 interacted with NbCAMTA2, a negative regulator of plant immunity. NbCAMTA2 was a negative regulator of NbHSP40 expression, and PsCRN108 could promote such inhibition activity of NbCAMTA2. Our results elucidated the multiple roles of PsCRN108 in the suppression of plant immunity and revealed a new mechanism by which the CRN effector hijacked transcription factors to affect immunity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
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Phytophthora , Phytophthora/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Choque Térmico/metabolismo , Inmunidad de la Planta , Enfermedades de las PlantasRESUMEN
Soil beneficial bacteria can effectively inhibit bacterial pathogens by assembling contact-dependent killing weapons, such as the type IVA secretion system (T4ASS). It's not clear whether these antibacterial weapons are involved in biotrophic microbial interactions in soil. Here we showed that an antifungal antibiotic 2,4-DAPG production of the soil bacterium, Pseudomonas protegens can be triggered by another soil bacterium, Lysobacter enzymogenes, via T4ASS by co-culturing on agar plates to mimic cell-to-cell contact. We demonstrated that the induced 2,4-DAPG production of P. protegens is achieved by intracellular detection of the T4ASS effector protein Le1519 translocated from L. enzymogenes. We defined Le1519 as LtaE (Lysobacter T4E triggering antifungal effects), which specifically stimulates the expression of 2,4-DAPG biosynthesis genes in P. protegens, thereby protecting soybean seedlings from infection by the fungus Rhizoctonia solani. We further found that LtaE directly bound to PhlF, a pathway-specific transcriptional repressor of the 2,4-DAPG biosynthesis, then activated the 2,4-DAPG production. Our results highlight a novel pattern of microbial interspecies and interkingdom interactions, providing a unique case for expanding the diversity of soil microbial interactions.
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Antifúngicos , Floroglucinol , Humanos , Antifúngicos/farmacología , Floroglucinol/metabolismo , Floroglucinol/farmacología , Hongos/metabolismo , Interacciones MicrobianasRESUMEN
Public metabolites such as vitamins play critical roles in maintaining the ecological functions of microbial community. However, the biochemical and physiological bases for fine-tuning of public metabolites in the microbiome remain poorly understood. Here, we examine the interactions between myxobacteria and Phytophthora sojae, an oomycete pathogen of soybean. We find that host plant and soil microbes complement P. sojae's auxotrophy for thiamine. Whereas, myxobacteria inhibits Phytophthora growth by a thiaminase I CcThi1 secreted into extracellular environment via outer membrane vesicles (OMVs). CcThi1 scavenges the required thiamine and thus arrests the thiamine sharing behavior of P. sojae from the supplier, which interferes with amino acid metabolism and expression of pathogenic effectors, probably leading to impairment of P. sojae growth and pathogenicity. Moreover, myxobacteria and CcThi1 are highly effective in regulating the thiamine levels in soil, which is correlated with the incidence of soybean Phytophthora root rot. Our findings unravel a novel ecological tactic employed by myxobacteria to maintain the interspecific equilibrium in soil microbial community.
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Myxococcales , Phytophthora , Glycine max , Tiamina , Rizosfera , VesículaRESUMEN
Elicitins are microbe-associated molecular patterns produced by oomycetes to elicit plant defense. It is still unclear whether elicitins derived from non-pathogenic oomycetes can be used as bioactive molecules for disease control. Here, for the first time we identify and characterize an elicitin named PpEli2 from the soil-borne oomycete Pythium periplocum, which is a non-pathogenic mycoparasite colonizing the root ecosystem of diverse plant species. Perceived by a novel cell surface receptor-like protein, REli, that is conserved in various plants (e.g. tomato, pepper, soybean), PpEli2 can induce hypersensitive response cell death and an immunity response in Nicotiana benthamiana. Meanwhile, PpEli2 enhances the interaction between REli and its co-receptor BAK1. The receptor-dependent immune response triggered by PpEli2 is able to protect various plant species against Phytophthora and fungal infections. Collectively, our work reveals the potential agricultural application of non-pathogenic elicitins and their receptors in conferring broad-spectrum resistance for plant protection.
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Transmembrane kinases (TMKs) are important mediators of cellular signaling cascades. The kinase domains of most metazoan and plant TMKs belong to the serine/threonine/tyrosine kinase (S/T/Y-kinase) superfamily. They share a common origin with prokaryotic kinases and have diversified into distinct subfamilies. Diverse members of the eukaryotic crown radiation such as amoebae, ciliates, and red and brown algae (grouped here under the umbrella term "protists") have long diverged from higher eukaryotes since their ancient common ancestry, making them ideal organisms for studying TMK evolution. Here, we developed an accurate and high-throughput pipeline to predict TMKomes in cellular organisms. Cross-kingdom analyses revealed distinct features of TMKomes in each grouping. Two-transmembrane histidine kinases constitute the main TMKomes of bacteria, while metazoans, plants, and most protists have a large proportion of single-pass TM S/T/Y-kinases. Phylogenetic analyses classified most protist S/T/Y-kinases into three clades, with clades II and III specifically expanded in amoebae and oomycetes, respectively. In contrast, clade I kinases were widespread in all protists examined here, and likely shared a common origin with other eukaryotic S/T/Y-kinases. Functional annotation further showed that most non-kinase domains were grouping-specific, suggesting that their recombination with the more conserved kinase domains led to the divergence of S/T/Y-kinases. However, we also found that protist leucine-rich repeat (LRR)- and G-protein-coupled receptor (GPCR)-type TMKs shared similar sensory domain architectures with respective plant and animal TMKs, despite that they belong to distinct kinase subfamilies. Collectively, our study revealed the functional diversity of TMKomes and the distinct origins of S/T/Y-kinases in protists.
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Plant cell-surface leucine-rich repeat receptor-like kinases (LRR-RLKs) and receptor-like proteins (LRR-RLPs) form dynamic complexes to receive a variety of extracellular signals. LRR-RLKs are also widespread in oomycete pathogens, whereas it remains enigmatic whether plant and oomycete LRR-RLKs could mediate cell-to-cell communications between pathogen and host. Here, we report that an LRR-RLK from the soybean root and stem rot pathogen Phytophthora sojae, PsRLK6, can activate typical pattern-triggered immunity in host soybean and nonhost tomato and Nicotiana benthamiana plants. PsRLK6 homologs are conserved in oomycetes and also exhibit immunity-inducing activity. A small region (LRR5-6) in the extracellular domain of PsRLK6 is sufficient to activate BAK1- and SOBIR1-dependent immune responses, suggesting that PsRLK6 is likely recognized by a plant LRR-RLP. Moreover, PsRLK6 is shown to be up-regulated during oospore maturation and essential for the oospore development of P. sojae. Our data provide a novel type of microbe-associated molecular pattern that functions in the sexual reproduction of oomycete, and a scenario in which a pathogen LRR-RLK could be sensed by a plant LRR-RLP to mount plant immunity.
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Phytophthora , Phytophthora/metabolismo , Plantas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Superficie Celular/metabolismo , Proteínas Tirosina Quinasas , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Soybean (Glycine max L. Merrill) is one of the most important economical crops. A large number of whole-genome resequencing datasets have been generated and are increasingly expanded for exploring genetic diversity and mining important quantitative trait loci. Most genome-wide association studies have focused on single-nucleotide polymorphisms, short insertions, and deletions. Nevertheless, structure variants mainly caused by transposon element mobilization are not fully considered. To fill this gap, we uniformly processed the publicly available whole-genome resequencing data from 5,521 soybean germplasm accessions and built an online soybean transposon insertion polymorphisms database named Soybean Transposon Insertion Polymorphisms Database (SoyTIPdb) (https://biotec.njau.edu.cn/soytipdb). The collected germplasm accessions derived from more than 45 countries and 160 regions representing the most comprehensive genetic diversity of soybean. SoyTIPdb implements easy-to-use query, analysis, and browse functions to help understand and find meaningful structural variations from TE insertions. In conclusion, SoyTIPdb is a valuable data resource and will help soybean breeders/researchers take advantage of the whole-genome sequencing datasets available in the public depositories.
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Estudio de Asociación del Genoma Completo , Glycine max , Glycine max/genética , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Análisis de Secuencia de ADN , Genoma de Planta/genéticaRESUMEN
Plant intracellular nucleotide-binding domain, leucine-rich repeat-containing receptors (NLRs) activate a robust immune response upon detection of pathogen effectors. How NLRs induce downstream immune defense genes remains poorly understood. The Mediator complex plays a central role in transducing signals from gene-specific transcription factors to the transcription machinery for gene transcription/activation. In this study, we demonstrate that MED10b and MED7 of the Mediator complex mediate jasmonate-dependent transcription repression, and coiled-coil NLRs (CNLs) in Solanaceae modulate MED10b/MED7 to activate immunity. Using the tomato CNL Sw-5b, which confers resistance to tospovirus, as a model, we found that the CC domain of Sw-5b directly interacts with MED10b. Knockout/down of MED10b and other subunits including MED7 of the middle module of Mediator activates plant defense against tospovirus. MED10b was found to directly interact with MED7, and MED7 directly interacts with JAZ proteins, which function as transcriptional repressors of jasmonic acid (JA) signaling. MED10b-MED7-JAZ together can strongly repress the expression of JA-responsive genes. The activated Sw-5b CC interferes with the interaction between MED10b and MED7, leading to the activation of JA-dependent defense signaling against tospovirus. Furthermore, we found that CC domains of various other CNLs including helper NLR NRCs from Solanaceae modulate MED10b/MED7 to activate defense against different pathogens. Together, our findings reveal that MED10b/MED7 serve as a previously unknown repressor of jasmonate-dependent transcription repression and are modulated by diverse CNLs in Solanaceae to activate the JA-specific defense pathways.
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Proteínas de Arabidopsis , Inmunidad de la Planta , Inmunidad de la Planta/genética , Ciclopentanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Proteobacteria primarily utilize acyl-homoserine lactones (AHLs) as quorum-sensing signals for intra-/interspecies communication to control pathogen infections. Enzymatic degradation of AHL represents the major quorum-quenching mechanism that has been developed as a promising approach to prevent bacterial infections. Here we identified a novel quorum-quenching mechanism revealed by an effector of the type IVA secretion system (T4ASS) in bacterial interspecies competition. We found that the soil antifungal bacterium Lysobacter enzymogenes OH11 (OH11) could use T4ASS to deliver the effector protein Le1288 into the cytoplasm of another soil microbiome bacterium Pseudomonas fluorescens 2P24 (2P24). Le1288 did not degrade AHL, whereas its delivery to strain 2P24 significantly impaired AHL production through binding to the AHL synthase PcoI. Therefore, we defined Le1288 as LqqE1 (Lysobacter quorum-quenching effector 1). Formation of the LqqE1-PcoI complex enabled LqqE1 to block the ability of PcoI to recognize/bind S-adenosy-L-methionine, a substrate required for AHL synthesis. This LqqE1-triggered interspecies quorum-quenching in bacteria seemed to be of key ecological significance, as it conferred strain OH11 a better competitive advantage in killing strain 2P24 via cell-to-cell contact. This novel quorum-quenching also appeared to be adopted by other T4ASS-production bacteria. Our findings suggest a novel quorum-quenching that occurred naturally in bacterial interspecies interactions within the soil microbiome by effector translocation. Finally, we presented two case studies showing the application potential of LqqE1 to block AHL signaling in the human pathogen Pseudomonas aeruginosa and the plant pathogen Ralstonia solanacearum.
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Pseudomonas fluorescens , Percepción de Quorum , Humanos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Acil-Butirolactonas/metabolismoRESUMEN
Phytophthora capsici is a notorious pathogen that infects various economically important plants and causes serious threats to agriculture worldwide. Plants deploy a variety of plant secondary metabolites to fend off pathogen attacks, but the molecular mechanisms are largely unknown. In this study, we screened 11 plant secondary metabolites to evaluate their biofumigation effects against P. capsici, and found that citral, carvacrol, and trans-2-decenal exhibited strong antimicrobial effects. Intriguingly, a low concentration of citral was effective in restricting P. capsici infection in Nicotiana benthamiana, but it was unable to inhibit the mycelial growth. A high concentration of citral affected the mycelial growth and morphology, zoospore germination, and cell membrane permeability of P. capsici. Further investigations showed that citral did not induce expression of tested plant immunity-related genes and reactive oxygen species (ROS) production, suggesting that a low concentration of citral could not trigger plant immunity. Moreover, RNA-Seq analysis showed that citral treatment regulated the expression of some P. capsici effector genes such as RxLR genes and P. cactorum-fragaria (PCF)/small cysteine-rich (SCR)74-like genes during the infection process, which was also verified by reverse transcription-quantitative PCR assay. Five candidate effector genes suppressed by citral significantly facilitated P. capsici infection in N. benthamiana or inhibited ROS triggered by flg22, suggesting that they were virulence factors of P. capsici. Together, our results revealed that plant-derived citral exhibited excellent inhibitory efficacy against P. capsici by suppressing vegetative growth and manipulating expression of effector genes, which provides a promising application of citral for controlling Phytophthora blight.
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Phytophthora , Virulencia/genética , Especies Reactivas de Oxígeno/metabolismo , Plantas , Nicotiana/genética , Enfermedades de las PlantasRESUMEN
The Phytophthora pathogen causes enormous damage to important agricultural plants. This group of filamentous pathogens is phylogenetically distant from fungi, making them difficult to control by most chemical fungicides. Lysobacter enzymogenes OH11 (OH11) is a biocontrol bacterium that secretes HSAF (Heat-Stable Antifungal Factor) as a broad-spectrum antifungal weapon. Here, we showed that OH11 could also control a variety of plant Phytophthora diseases caused by three major oomycetes (P. sojae, P. capsici and P. infestans). We provided abundant evidence to prove that OH11 protected host plants from Phytophthora pathogen infection by inhibiting mycelial growth, digesting cysts, suppressing cyst germination, and eliciting plant immune responses. Interestingly, the former two processes required the presence of HSAF, while the latter two did not. This suggested that L. enzymogenes could prevent Phytophthora infection via multiple previously unknown mechanisms. Therefore, this study showed that L. enzymogenes could serve as a promising alternative resource for promoting plant resistance to multiple Phytophthora pathogens.
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Walnut is a natural source of antioxidants. Its antioxidant capacity is determined by the distribution and composition of phenolics. The key phenolic antioxidants in various forms (free, esterified, and bound) in walnut kernel (particularly seed skin) are unknown. The phenolic compounds in twelve walnut cultivars were analyzed using ultra-performance liquid chromatography coupled with a triple quadrupole mass spectrometer in this study. A boosted regression tree analysis was used to identify the key antioxidants. Ellagic acid, gallic acid, catechin, ferulic acid, and epicatechin were abundant in the kernel and skin. The majority of phenolic acids were widely distributed in the free, esterified, and bound forms in the kernel but more concentrated in bound phenolics in the skin. The total phenolic levels of the three forms were positively correlated with antioxidant activities (R = 0.76-0.94, p < 0.05). Ellagic acid was the most important antioxidant in the kernel, accounting for more than 20%, 40%, and 15% of antioxidants, respectively. Caffeic acid was responsible for up to 25% of free phenolics and 40% of esterified phenolics in the skin. The differences in the antioxidant activity between the cultivars were explained by the total phenolics and key antioxidants. The identification of key antioxidants is critical for new walnut industrial applications and functional food design in food chemistry.
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SUMMARY: Fungi form a large and heterogeneous group of eukaryotic organisms with diverse ecological niches. The high importance of fungi contrasts with our limited understanding of fungal lifestyle and adaptability to environment. Over the last decade, the high-throughput sequencing technology produced tremendous RNA-sequencing (RNA-seq) data. However, there is no comprehensive database for mycologists to conveniently explore fungal gene expression and alternative splicing. Here, we have developed FungiExp, an online database including 35 821 curated RNA-seq samples derived from 220 fungal species, together with gene expression and alternative splicing profiles. It allows users to query and visualize gene expression and alternative splicing in the collected RNA-seq samples. Furthermore, FungiExp contains several online analysis tools, such as differential/specific, co-expression network and cross-species gene expression conservation analysis. Through these tools, users can obtain new insights by re-analyzing public RNA-seq data or upload personal data to co-analyze with public RNA-seq data. AVAILABILITY AND IMPLEMENTATION: The FungiExp is freely available at https://bioinfo.njau.edu.cn/fungiExp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Empalme Alternativo , ARN , Análisis de Secuencia de ARN , Perfilación de la Expresión Génica , Expresión Génica , Programas InformáticosRESUMEN
Yam (Dioscorea spp.) is an important source of food and energy in the world, and is also widely cultivated in China (Frossard et al. 2017). Yam is rich in fiber and antioxidants, and can significantly reduce blood sugar. Therefore, it is also known as a medicinal crop with high medicinal value in China (Cao et al. 2021). In October 2021, leaf spots were observed on the seven month old yam leaves of a commercial yam field in Nanjing city, Jiangsu Province, China. The field had a disease severity of approximately 25% and an incidence of 30%, and the infected plants displayed poor development. Symptoms in the leaves included irregular yellow to brown spots of different sizes, and lesion spots later turned dark brown, resulting in necrotic leaves (Fig. S1A). Four symptomatic leaves collected from 3 yam plants were rinsed with water, cut into 2-mm small square leaf pieces and were surface-sterilized in 70% ethanol and 2% sodium hypochlorite for 2 min, then rinsed three times with sterile distilled water. The sterilized leaf tissues were plated onto potato dextrose agar (PDA) containing ampicillin and rifampicin, and then were incubated at 25°C in the dark for 3-5 days. Three isolates (NAU-1, NAU-2, and NAU-3) were obtained from a total of four leaf samples by purifying of single-spore culture. Nine agar plugs (6×6 mm2) were transferred into 8 mL of PDA in a 70 mm plate at 25°C to observe colony morphology. The three isolates had identical morphological features. For the representative isolate NAU-1, colonies were white and fluffy in appearance during initial 4 days, and became gray-brown with the onset of sporulation after 5 days (Fig. S1B). Microscopic observation showed that mycelia were smooth, branched, and septate (Fig. S1C) and conidia were single-celled, black, spherical to subspherical, measuring 10.7 ± 0.9 µm × 12.5 ± 2.6 µm in diameter (n=50) (Fig. S1D). Morphological features suggested that these isolates possessed the same characteristics. For accurate identification, the genomic DNA was extracted from each isolate using Fungi Genomic DNA Extraction Kit (D2300; Solarbio, Beijing, China). The internal transcribed spacer (ITS) sequence, ß-tubulin (TUB2) gene, and translation elongation factor 1-alpha (Tef-1α) gene, were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and EF1/EF2 (O'Donnell et al. 1998), respectively. These sequences were deposited in GenBank (ITS: ON394529, OP735574, and OP735562; TUB2: ON427830, OP756526, and OP756528; Tef-1α: ON427831, OP756525, and OP756527 for NAU-1, NAU-2, and NAU-3, respectively). The ITS, TUB2, and Tef-1α sequences of three isolates showed high similarity (>98.5%) to the corresponding sequences (MT732051.1, KY019554.1, and KY019413.1) of Nigrospora oryzae in GenBank. Further phylogenetic analysis showed that the three isolates clustered with N. oryzae (Fig. S2). Therefore, based on morphological and molecular evidence, the three isolates were identified as N. oryzae. N. oryzae has been reported to cause leaf spot on cotton and ginger in China (Zhang et al. 2012; Liu et al. 2022). Furthermore, the pathogenicity of isolated N. oryzae was tested on healthy, potted 1-year-old yam plants. The 6-mm agar plugs-containing mycelia from 4-day-old PDA cultures and agar blocks (control) were used to inoculate sixteen wounded yam leaves, followed by maintaining in a growth chamber under 16 h day at 25°C and 8 h night at 20°C. After six days post inoculation, all the inoculated yam leaves exhibited similar symptoms observed in the field, whereas the uninoculated leaves remained symptomless (Fig. S1E). The experiments were repeated three times with similar results. N. oryzae was reisolated from the infected plants and confirmed to be the same pathogen by using morphological and molecular methods. To our knowledge, this is the first report of N. oryzae causing leaf spot disease of yams in China. Identification of this pathogen would assist in developing strategies to efficiently control the spread of the disease on Chinese yams.
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Loss of flagellar genes causes a nonmotile phenotype. The genus Lysobacter consists of numerous environmentally ubiquitous, nonflagellated bacteria, including Lysobacter enzymogenes, an antifungal bacterium that is beneficial to plants. L. enzymogenes still has many flagellar genes on its genome, although this bacterium does not engage in flagella-driven motility. Here, we report that loss of certain flagellar genes allows L. enzymogenes to strengthen its evolutionarily gained capacity in fungal killing. To clarify why this bacterium loses flagellar genes during the evolutionary process, we cloned several representative flagellar genes from Xanthomonas oryzae, a flagellated, phylogenetically related species of Lysobacter, and introduced them individually into L. enzymogenes to mimic genomic reacquisition of lost flagellar genes. Heterogeneous expression of the three X. oryzae flagellar structural genes (Xo-motA, Xo-motB, Xo-fliE) and one flagellar regulatory gene (Xo-fleQ) remarkably weakened the bacterial capacity to kill fungal pathogens by impairing the synthesis of an antifungal weapon, known as the heat-stable antifungal factor (HSAF). We further investigated the underlying mechanism by selecting Xo-FleQ as the representative because it is a master transcription factor responsible for flagellar gene expression. Xo-FleQ inhibited the transcription of operon genes responsible for HSAF synthesis via direct binding of Xo-FleQ to the promoter region, thereby decreasing HSAF biosynthesis by L. enzymogenes. These observations suggest a possible genome and function coevolution event, in which an antifungal bacterium deletes certain flagellar genes in order to enhance its ability to kill fungi. IMPORTANCE It is generally recognized that flagellar genes are commonly responsible for the flagella-driven bacterial motility. Thus, finding nonflagellated bacteria partially or fully lost flagellar genes is not a surprise. However, the present study provides new insights into this common idea. We found that loss of either certain flagellar structural or regulatory genes (such as motA, motB, fliE, and fleQ) allows a nonflagellated, antifungal bacterium (L. enzymogenes) to stimulate its fungal-killing capacity, outlining a genome-function coevolution event, where an antifungal bacterium "smartly" designed its genome to "delete" crucial flagellar genes to coordinate flagellar loss and fungal predation. This unusual finding might trigger bacteriologists to reconsider previously ignored functions of the lost flagellar genes in any nonflagellated, pathogenic, or beneficial bacteria.