Microbiological Research

Ammonia opens trap formation in the nematode-trapping (NT) fungus Arthrobotrys oligospora, an intriguing morphological switch in NT fungi, where saprophytic mycelia are converted to pathogenic organs. Endocytosis plays a prominent role in nutrient uptake, signaling cascades, and maintenance of cellular homeostasis in higher eukaryotes. Here, we demonstrate that ammonia efficiently promotes endocytosis via the formation of 3D-adhesive mycelial nets in A. oligospora. Trap production is followed by the presence of massive multivesicular bodies (MVBs) and membrane rupture and repair. Additionally, both the ubiquitin-proteasome system and the endosomal sorting complex for transport (ESCRT) pathway are immediately linked to endocytosis regulation and MVB formation in ammonia-induced trap formation. Moreover, disruption of the ESCRT-1 complex subunit proteins AoHse and AoVps27 led to the complete loss of membrane endocytosis and trap formation. Finally, the deletion of the deubiquitinase AoSst2 caused a significant reduction in the number of trap structures produced in response to exposure to ammonia or nematodes. Overall, our results increase our knowledge of the molecular mechanisms underlying the phenotypic changes in the NT fungal group, demonstrating that the endocytosis-ESCRT-MVB pathway participates in the regulation of trapping organs. Author SummaryThe lifestyle switch of nematode-trapping (NT) fungi is a significant event that increases their pathogenicity to nematode prey, which has resulted in large losses to agricultural crops worldwide. Here, we describe the molecular mechanism underlying how this fungal group forms a NT structure in response to ammonia, a widely preferred nitrogen source in soil niches. Ammonia enhances the endocytosis process, ubiquitin-proteasome system, and endosomal sorting complex for transport (ESCRT) pathway of the model NT fungus A. oligospora, thereby generating enriched multivesicular bodies (MVBs) during trap formation. In this process, the cell membrane morphology is remarkably damaged and then repaired. We further found that disruption of the ESCRT-0 subcomplex or ubiquitinase severely blocked trap production and membrane reorganization. Our study provides a new understanding of endocytosis-ESCRT-MVB flux in the transition of fungal NT organs.

Fusarium sacchari is one of the most important sugarcane pathogens that causes Pokkah boeng disease (PBD) in China. 14-3-3 proteins have been shown to play vital roles in developmental processes in dimorphic transition, signal transduction and carbon metabolism in some phytopathogenic fungi, but were poorly understood in F. sacchari. In this study, two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2 in F. sacchari, were investigated. Although both FsBmh1 and FsBmh2 were expressed at vegetative growth stage, FsBmh1 was repressed at sporulation stage in vitro. In order to clarify the roles of FsBmh1 and FsBmh2, deletion mutants {Delta}FsBmh1 and {Delta}FsBmh2 were constructed. Phenotypic defects, including hyphal branching, hyphal septation, conidiation, spore germination and colony growth, were more severe in {Delta}FsBmh2 than in {Delta}FsBmh1, although virulence attenuation was observed in both mutants. To further explore the relationship between FsBmh1 and FsBmh2, the combination of deletion and silencing ({Delta}FsBmh/sFsBmh) and overexpression (O-FsBmh) mutants were constructed and characterized. Compared to the single allele deletion, combinations of {Delta}FsBmh1/sFsBmh2 or {Delta}FsBmh2/sFsBmh1 showed more severe manifestations in general, suggesting a redundancy in function of the two 14-3-3 genes. Comparative transcriptome analysis revealed that more functional genes were affected in {Delta}FsBmh2 than in {Delta}FsBmh1. Redundancy in function between FsBmh1 and FsBmh2 suggests that 14-3-3 is vitally important for the organism and distinction in roles between the two isoforms may be resulted from the divergence in evolution. To the best of our knowledge, this was the first report on the distinct roles of 14-3-3 protein isoforms in a pathogenic fungus. Knowledge gained from this study should be of help to further understand the regulation mechanism of pathogenicity-related traits in fungal pathogens and for the development of new strategy for control of PBD in particular.

Biotrophic and hemibiotrophic fungi have evolved the ability to colonize living plant cells, but how they establish biotrophic growth by remodeling gene expression is poorly understood. By using in planta invasive hyphae (IH) of Magnaporthe oryzae to perform an integrated Chromatin immunoprecipitation sequencing (ChIPseq) and RNA-seq analysis, combining with biological and cellular analyses, we found Polycomb repressive complex 2 (PRC2)-mediated epigenetic repression plays a key role in regulating biotrophic growth. ChIPseq for biotrophic IH samples identified 1701 PRC2 target genes. RNA-seq analysis showed that expression of 462 PRC2 target genes were up-regulated in the {Delta}suz12 mutant, while 82 were down-regulated, indicating a major role of PRC2 in gene repression of IH. During biotrophic growth, PRC2 repressed fungal cell wall synthesis genes and extracellular enzyme genes required for penetration, and secondary metabolites biosynthesis genes required for necrotrophic growth. A great number of effector-encoding genes were repressed by PRC2, which were highly expressed during penetration stage, suggesting PRC2 coordinates biotrophic growth by regulating effector suppression for immune evasion. This regulation was finely coordinated by Pmk1, through regulating phosphorylation, nuclear localization and protein abundance of Suz12. Our results indicate that the Pmk1-PRC2 regulatory module is required for gene remodeling to facilitate biotrophic growth in M. oryzae. IMPORTANCEBiotrophic and hemibiotrophic fungi establish a biotrophic stage for infection in host cells. For example, M. oryzae forms appressoria to penetrate host cell and establish a biotrophic growth stage for infection. How gene expression patterns are elaborately controlled for fungal biotrophic growth is largely unknown. In this study, we found that, the PRC2-mediated H3K27me3 repressed fungal penetration-required cell wall synthesis genes and extracellular enzyme genes, and necrotrophic growth-required secondary metabolites biosynthesis genes for biotrophic growth. Interestingly, a great number of effector-encoding genes were also repressed by PRC2 at biotrophic stage, which were highly expressed at penetration stage, suggesting PRC2 coordinates biotrophic growth by regulating effector suppression for immune evasion. The PRC2-mediated epigenetic repression is therefore required for the gene expression remodeling during fungal infection. This regulation was finely coordinated by Pmk1, through regulating nuclear localization and protein abundance of the PRC2 component Suz12.

Recent comparative genomics and mechanistic analyses support the existence of a fungal immune system. Fungi encode genes with features similar to non-self recognition systems in plants, animals, and bacteria. However, limited functional or mechanistic evidence exists for the surveillance-system recognition of heterologous microbes in fungi. We found that Neurospora species coexist with Pseudomonas in their natural environment. We leveraged two model organisms, Neurospora crassa and Pseudomonas syringae DC3000 (PSTDC3000) to observe immediate fungal responses to bacteria. PSTDC3000 preferentially surrounds N. crassa cells on a solid surface, causing environmental dependent growth responses, bacterial proliferation and varying fungal fitness. Specifically, the Type III secretion system (T3SS) {Delta}hrcC mutant of PSTDC3000 colonized N. crassa hyphae less well. To dissect initial cellular signaling events within the population of germinated asexual spores (germlings), we performed transcriptomics on N. crassa after PSTDC3000 inoculation. Upon contact with live bacteria, a subpopulation of fungal germlings initiate a response as early as ten minutes post-contact revealing transcriptional differentiation of Reactive Oxygen Species (ROS) mechanisms, trace metal warfare, cell wall remodeling dynamics, multidrug-efflux transporters, secondary metabolite synthesis, and excretion. We dissected mutants of plausible receptors, signaling pathways, and responses that N. crassa uses to detect and mount a defense against PSTDC3000 and found seven genes that influence resistant and susceptibility phenotypes of N. crassa to bacterial colonization. Mutants in genes encoding a ctr copper transporter (tcu-1), ferric reductase (fer-1), superoxide reductase (sod-2), multidrug resistance transporter (mdr-6), a secreted lysozyme-Glycoside hydrolase (lyz) and the Woronin body tether leashin (NCU02793, lah-1 and lah-2) showed a significant reduction of growth in the presence of bacteria, allowing the bacteria to fully take over the fungal mycelium faster than wildtype. In this study we provide a bacterial-fungal model system within Dikarya that allows us to begin to dissect signaling pathways of the putative fungal immune system.

Fungal innate immunity resembles mammalian innate immunity. It does not employ toll-like receptors (TLRs), but should employ endocytosis of non-fungal molecular patterns recognized by nuclear-localizing receptors (NLR). Downstream, both types of receptors are Mammalian Ste20 kinases (MSTs). We identified an MST3 ortholog in the plant pathogens Fusarium graminearum (FgMST3) and Magnaporthe oryzae (MoMST3). We knocked out both genes and investigated mutants using a standard panel of tests for growth, development, and pathogenicity for the respective fungi. Both{Delta} FgMST3 and{Delta} MoMST3 strains showed reduced pathogenicity. The deletions negatively affected conidia production and conidia germination but had little effect on growth rate. However, the two mutants reacted differently to various stress treatments, especially to Zn2+ and gentamicin. In addition, we constructed an innate immunity reporter system for F. graminearum to detect less than 4-hour responses to non-self-molecular patterns (NSMP) like bacterial outer membrane vesicles (OMVs) and trace levels of sucrose, indicating plant. The reporter gene responses to OMVs of MST3 mutant strains are severely reduced. Our results indicate that both MoMst3 and FgMst3 are involved in fungal innate immunity downstream of unknown NLR proteins, motivating studies to identify genes for the NLR receptors. Finding such and investigating how they work and vary between fungal species and strains should be essential for understanding fungal biotic interactions with viruses, bacteria, plants, and animals.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.

Isoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Through IPT adaptation on PDA medium, we obtained a variety of IPT-resistant mutants. Based on their EC50 values to IPT, the resistant mutants were mainly divided into three distinct categories i.e., low resistance (LR, 6.5 [&le;] EC50 < 13.0 g/mL), moderate resistance 1 (MR-1, 13.0 [&le;] EC50 < 25.0 g/mL), and moderate resistance 2 (MR-2, 25.0 [&le;] EC50 < 35.0 g/mL). Molecular analysis of MoIRR (Magnaporthe oryzae isoprothiolane resistance related) gene demonstrated that it was associated only with the moderate resistance in MR-2 mutants, indicating that other mechanisms were associated with resistance in LR and MR-1 mutants. In this study, we mainly focused on the characterization of low resistance to IPT in M. oryzae. Mycelial growth and conidial germination were significantly reduced, indicating fitness penalties in LR mutants. Based on the differences of whole genome sequences between parental isolate and LR mutants, we identified a conserved MoVelB gene, encoding the velvet family transcription factor, and genetic transformation of wild type isolate verified that MoVelB gene was associated with the low resistance. Based on molecular analysis, we further demonstrated that the velvet family proteins VelB and VeA were indispensable for IPT toxicity and the deformation of the LaeA-VeA-VelB heterotrimer played a vital role for the low IPT-resistance in M. oryzae, most likely through the down-regulation of the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT. Author summaryIsoprothiolane (IPT) resistance has emerged in Magnaporthe oryzae, due to the long-term usage of IPT to control rice blast in China, yet the mechanisms of the resistance remain largely unknown. Here, we explored the mechanisms of low IPT resistance in M. oryzae. Combining the whole genome sequencing and genetic transformation, we identified a conserved MoVelB gene, encoding the velvet family transcription factor to be a determinant for IPT toxicity. We further demonstrated that the deformation of the LaeA-VeA-VelB heterotrimer conferred the low IPT-resistance in M. oryzae, most likely through down-regulating the secondary metabolism-related genes or CYP450 genes to reduce the toxicity of IPT. This study improved our understanding of the resistance mechanism as well as the mode of action of IPT which will be helpful for making suitable strategies to manage the emerging resistance of IPT in M. oryzae.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate protein-coding gene expression primarily found in plants and animals. Fungi produce microRNA-like RNAs (milRNAs) that are structurally similar to miRNAs and functionally important in various biological processes. The fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. It remains uncharacterized about the biosynthesis and functions of milRNAs in Foc. In this study, we investigated the biological function of milRNAs contributing to Foc pathogenesis. Within 24 hours post infecting the host, the Argonaute coding gene FoQDE2, and two Dicer coding genes FoDCL1 and FoDCL2, all of which are involved in milRNA biosynthesis, were significantly induced. FoQDE2 deletion mutant exhibited decreased virulence and hypersensitivity to hydrogen peroxide (H2O2). These results indicate that milRNA biosynthesis is crucial for Foc pathogenesis. By small RNA sequencing, we identified 364 small RNA-producing loci in the Foc genome, 25 of which were significantly downregulated in the FoQDE2 deletion mutant, from which milR-87 was verified as a FoQDE2-depedent milRNA based on qRT-PCR analysis. Through deletion and overexpression of milR-87 in the wild-type Foc strain, functions of milR-87 were studied. The results showed that milR-87 is crucial for Foc virulence in infection process. We furthermore identified a glycosyl hydrolase-coding gene, FOIG_15013, as the direct target of milR-87. The FOIG_15013 deletion mutant displayed a dramatic increase in the growth, conidiation and virulence. Transient expression of FOIG_15013 in Nicotiana benthamiana leaves activates the host defense responses. Collectively, this study documents the involvement of milRNAs in the manifestation of the devastating fungal disease in banana, and demonstrates the importance of milRNAs in the pathogenesis and other biological processes. Further analyses of the biosynthesis and expression regulation of fungal milRNAs may offer a novel strategy to combat devastating fungal diseases. Author summaryThe fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of Panama disease that threatens global banana production. As a typical representative of F. oxysporum species complex, the pathogen has been widely concerned. However, pathogenesis of Foc is not fully elucidated. In particular, pathogenic regulatory mechanism of the microRNA like small RNAs (milRNAs) found in Foc is unknown. Here, we found that FoQDE2, one Argonaute coding gene, and two Dicer coding genes FoDCL1 and FoDCL2, which are involved in milRNA biosynthesis, are significantly induced during the early infection stage of Foc. The results suggested that the milRNAs biosynthesis mediated by these genes may play an active role in the infection process of Foc. Based on this assumption, we subsequently found a FoQDE2-dependent milRNA (milR-87) and identified its target gene. Functional analysis showed that FoQDE2, miR-87 and its target gene were involved in the pathogenicity of Foc in different degree. The studies help us gain insight into the pathogenesis with FoQDE2, milR-87, and its target gene as central axis in Foc. The identified pathogenicity-involved milRNA provides an active target for developing novel and efficient biocontrol agents against Panama disease.

Maize seedling blight, caused by the phytopathogenic fungus Fusarium verticillioides, is a common and rapidly spreading disease that negatively impacts grain quality and productivity. Control of this pathogen is complicated by its complex infection process and the tendency for resistance to conventional chemical pesticides. The use of biological control agents has been recognized as an environmentally friendly and sustainable solution for the control of plant diseases. In our study, we isolated and identified Paenibacillus peoriae 3-B4 from maize leaves, which exhibited an inhibition rate of 59.92% against F. verticillioides in greenhouse experiments. Genome sequencing of P. peoriae 3-B4 revealed a chromosome of 5,912,131 bp, featuring a GC content of 45.51% on average and 5383 annotated coding sequences. Eight gene clusters associated with secondary metabolites with antifungal activity and thirteen genes associated with induced systemic resistance and pattern-triggered immunity were identified. Transcriptomic analysis identified 8,997 differentially expressed maize genes, with key defense genes (e.g., NPR1, bZIP, MYB, LRR, WRKY) enriched in MAPK signaling and plant-pathogen interactions. 16S rRNA analysis showed significant shifts in microbial communities, particularly with an increase in the abundance of beneficial genera like Paenibacillus, Delftia, and Corynebacterium. The combined analysis of differentially expressed genes and microbial communities indicated that they synergistically enhance pathogen resistance in maize. Our findings outline the potential mechanisms by which P. peoriae 3-B4 inhibits F. verticillioides infection and open possibilities of exploiting biological control strategies to control maize seedling blight.

Many plant-associated bacteria produce plant- mimicking hormones which are involved in modulating host physiology. However, their function in modulating bacterial physiology has not been reported. Here we show that the XopQ protein, a type-III effector of the rice pathogen, Xanthomonas oryzae pv. oryzae (Xoo), is involved in cytokinin biosynthesis. Xoo produces and secretes an active form of cytokinin which enables the bacterium to maintain a planktonic lifestyle and promotes virulence. RNA-seq analysis indicates that the cytokinin produced by Xoo is required for the regulation of several genes which are involved in biofilm formation. We have also identified the Xoo isopentenyl transferase gene, which is involved in the cytokinin biosynthesis pathway and is required for maintaining planktonic behaviour and virulence. Furthermore, mutations in the predicted cytokinin receptor kinase (PcrK) and the downstream response regulator (PcrR) of Xoo phenocopy the cytokinin biosynthetic mutants, but are not complemented by supplementation with exogenous cytokinin. Cytokinin biosynthetic functions are encoded in a number of diverse bacterial genomes suggesting that cytokinin may be a widespread signalling molecule in the bacterial kingdom.

Bacterial-fungal interactions (BFIs) are important in ecosystem dynamics, especially within the soil rhizosphere. The bacterium Bacillus velezensis SQR9 and the fungus Trichoderma guizhouense NJAU 4742 have garnered considerable attention due to their roles in promoting plant growth and protecting their host against pathogens. In this study, we utilized these two model microorganisms to investigate BFI. We firstly demonstrate that while co-inoculation of B. velezensis and T. guizhouense could promote tomato growth, these two microorganisms display mutual antagonism on agar solidified medium. To resolve this contradiction, we developed an inoculation method, that allows B. velezensis colonization of T. guizhouense hyphae and performed a transcriptome analysis. During colonization of the fungal hyphae, B. velezensis SQR9 upregulates expression of biofilm related genes (e.g. eps, tasA, and bslA) that is distinct from free-living cells. This result suggested an intricate association between extracellular matrix expression and hyphae colonization. In accordance, deletion epsD, tasA, or both epsD and tasA genes of B. velezensis diminished colonization of the T. guizhouense hyphae. The insights from our study demonstrate that soil BFIs are more complex than we understood, potentially involving both competition and cooperation. These intricate biofilm-mediated BFI dynamics might contribute to the remarkable diversity observed within soil microbiota, providing a fresh perspective for further exploration of BFIs in the plant rhizosphere.

Sporulation is the most widespread means of reproduction and dispersal in fungi. In the Basidiomycota, sexual spores are produced on specialised cells known as basidia, from which they are discharged forcibly by a powered process called ballistospory, the highest known acceleration in nature. However, the genetics of sporulation, in particular postmeiotic events related to spore morphogenesis and ballistospory, remain poorly known. Here, we explore the genetics of these processes, based on a new, highly conserved transcription factor, Sporulation-Related Regulator 1 (SRR1), and its putative downstream regulatory network. Reverse genetics of Srr1 in the model mushroom Coprinopsis cinerea and commercially produced oyster mushroom indicated a conserved role of Srr1 in sporulation across Agaricomycetes. RNA-Seq analysis and motif-based inference of a hypothetical SRR1 gene regulatory network allowed delimiting putative targets regulated by SRR1 in a direct and indirect manner. Using this network and comparative genomics, we identified genes associated with ballistospory, including a putative SRR1-target chitinase, which was found to be required for normal spore production and morphology. Overall, our study offers new insights into the genetic mechanisms governing postmeiotic spore morphogenesis and ballistospory in the Agaricomycetes.

Plants provide distinct ecological niches for diverse microbial communities, with each member adopting strategies tailored to the specific ecological niche it inhabits. Two foliar niches, the leaf surface (epiphytic environment) and the apoplast, impose distinct physiological constraints on microbial fitness, particularly for hemibiotrophic pathogens. In this study, we investigated how these environments shape the transcriptional responses of Xanthomonas perforans (Xp), a tomato pathogen, and how its virulence factors, metabolic pathways, and regulatory networks are spatially and temporally coordinated during disease progression. Transcriptome profiling of a pathogen recovered from the leaf surface and apoplast revealed pronounced niche-specific and colonization stage-specific gene expression patterns. Early epiphytic colonization was characterized by activation of chemosensing, and motility pathways that facilitate pathogen relocation and acquisition of limiting nutrients such as iron and phosphate. This stage also featured induction of DNA and protein repair systems, quorum sensing pathways, phenylalanine degradation and tyrosine conversion to counter phenylpropanoid defenses, genes involved in mitigating osmotic and oxidative stress, active DNA exchange machinery, and type VI secretion system-mediated microbial competition. Upon entry into the apoplast, Xp shifted toward active metabolism and replication, accompanied by investment in type II and III secreted virulence factor expression. Genes involved in evasion of plant immunity and overcoming of host-mediated nutrient sequestration were also upregulated, including those involved in quinone detoxification, phosphate and sulfur uptake, and fatty acid, xanthan, and LPS biosynthesis. During late apoplastic colonization, the pathogen transitioned again towards strong stress response activation, followed by renewed expression of flagellar motility and chemotaxis genes, suggesting preparation for dissemination. Notably, genes associated with oxidative and nutrient stress were enriched across both niches, although specific components differed. Type IV pili, conjugation genes, and plasmid-borne type III effectors were induced early in both niches, suggesting their niche-independent role in initial establishment. Together, these findings reveal a coordinated spatio-temporal regulatory strategy during the transition from the leaf surface to the apoplast. Author SummaryXanthomonas perforans is a foliar bacterial pathogen that infects tomato plants and leads to severe yield losses. To establish a successful infection, the pathogen must overcome a series of environmental and host-imposed challenges. This study characterizes the traits activated at distinct stages of infection, during both early and late pathogenesis, and across different niches, including the leaf surface and its interior (apoplastic) space. On the leaf surface Xanthomonas mainly focuses on movement, communication, and survival against stress and starvation with the major functions related to motility, nutrient uptake, and DNA transfer during early stages. Once inside the leaf, the bacteria switches tactics to focus primarily on reproduction, defense against the plant immune response, production of factors that weaken the plants defenses and gaining access to nutrients the plant normally restricts. Understanding the different stages of infection may inform how the crosstalk among host and pathogen unfolds during pathogenesis allowing us to understand the host environment. These findings can help us discover pathogen weaknesses that could be targeted for disease management.

Pseudomonas aeruginosa is the major infectious agent among Gram-negative bacteria which causes both acute and chronic infections without any tissue specificity. Infections due to P. aeruginosa are hard to treat, as it entails various strategies like virulence factors synthesis, drug efflux systems & resistance and protein secretion systems during pathogenesis. Despite extensive research in Pseudomonas pathogenesis, novel drug targets and potential therapeutic strategies are inevitable. In this study, we investigated the genetic requirements of P.aeruginosa PAO1 for rat cardiomyocyte (H9C2) infection by insertion sequencing (INSeq). A mutant library comprising ~70,000 mutants of PAO1 was generated and the differentiated form of H9C2 cells (d-H9C2) was infected with the library. The infected d-H9C2 cells were maintained with antibiotic-protection and without any antibiotics in the growth media for 24 h. Subsequently, DNA library for INSeq was prepared, sequenced and fitness analysis was performed. A-One hundred and thirteen mutants were negatively selected in the infection condition with antibiotic-protection, whereas 143 mutants were negatively selected in antibiotic-free condition. Surprisingly, a higher number of mutants showed enriched fitness than the mutants of reduced fitness during the infection. We demonstrated that the genes associated with flagella and T3SS are important for adhesion and invasion of cardiomyocytes, while pili and proteases are conditionally essential during host cell lysis. Take away{checkmark} Fitness of P.aeruginosa mutants were analyzed during cardiomyocyte infection {checkmark}Genes involve amino acid transport & metabolism and signal transduction are important during intracellular lifestyle {checkmark}OMVs play a crucial role during infection and pathogenesis {checkmark}Flagella and T3SS are conditionally essential for adhesion and invasion, whereas pili and proteases are conditionally essential during host cell lysis

Sexual and asexual reproduction is ubiquitous in eukaryotes. PI3K/AKT signaling pathway can modulate sexual reproduction in mammals. However, this signaling pathway modulating sexual and asexual reproduction in fungi is scarcely understood. SeASF1, a SeH4 chaperone, could manipulate sexual and asexual reproduction of Stemphylium eturmiunum. SeDJ-1, screened from Se{Delta}asf1 transcriptome, was confirmed to regulate sexual and asexual development by RNAi, of which the mechanism was demonstrated by detecting transcriptional levels and protein interactions of SeASF1, SeH4 and SeDJ-1 by qRT-PCR, and Y2H, Co-IP and Pull-down, respectively. SeASF1 coupling SeH4 bound SeDJ-1 to arouse the sexual and asexual activity. In S. eturmiunum genome, SeDJ-1 was upstream while SeGSK3 was downstream in PI3K/AKT signaling pathway. Moreover, SeDJ-1 interacted with SePI3K or SeGSK3 in vivo and in vitro. Significantly, SeDJ-1 or SePI3K could effectively stimulate sexual activity alone, but SePI3K could recover the sexual development of SiSeDJ-1. Meanwhile, SeDJ-1-M6 was a critical segment for interaction of SeDJ-1 with SePI3K. SeDJ-1-M6 played a critical role in irritating sexual reproduction in SiSePI3K, which further uncovered the regulated mechanism of SeDJ-1. Summarily, SeASF1 coupling SeH4 motivates SeDJ-1 to arouse SePI3K involved in sexual reproduction. Thus, SeASF1 can activate PI3K/AKT signaling pathway to regulate sexual and asexual development in filamentous ascomycete.

Colletotrichum fungi infect a wide diversity of monocot and eudicot hosts, causing plant diseases on almost all economically important crops worldwide. In addition to its economic impact, Colletotrichum is a suitable model for the study of gene family evolution on a fine scale to uncover events in the genome that are associated with the evolution of biological characters important for host interactions. Here we present the genome sequences of 30 Colletotrichum species, 18 of them newly sequenced, covering the taxonomic diversity within the genus. A time-calibrated tree revealed that the Colletotrichum ancestor diverged in the late Cretaceous around 70 million years ago (mya) in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from eudicots to monocots during the evolution of this pathogen, coinciding with a progressive shrinking of the degradative arsenal and expansions in lineage specific genes. Comparative transcriptomics of four reference species with different evolutionary histories and adapted to different hosts revealed similarity in gene content but differences in the modulation of their transcription profiles. Only a few orthologs show similar expression profiles on different plant cell walls. Combining genome sequences and expression profiles we identified a set of core genes, such as specific transcription factors, involved in plant cell wall degradation in Colletotrichum.Together, these results indicate that the ancestral Colletotrichum were associated with eudicot plants and certain branches progressively adapted to different monocot hosts, reshaping part of the degradative and transcriptional arsenal.

The E. coli strain harboring the polyketide synthase (Pks) island encodes the genotoxin colibactin, a secondary metabolite reported to have severe implications for human health and for the progression of colorectal cancer. The present study involved whole-genome-wide comparison and phylogenetic analysis of pks harboring E. coli isolates to gain insight into the distribution and evolution of these organism. Fifteen E. coli strains isolated from patients with ulcerative colitis were sequenced, 13 of which harbored pks islands. In addition, 2,654 genomes from the public database were also screened for pks harboring E. coli genomes, 158 of which were pks-positive isolates. Whole-genome-wide comparison and phylogenetic analysis revealed that 171 (158+13) pks-positive isolates belonged to phylogroup B2, and most of the isolates associated to sequence types ST73 and ST95. One isolate from an ulcerative colitis (UC) patient was of the sequence type ST8303. The maximum likelihood tree based on the core genome of pks-positive isolates revealed horizontal gene transfer across sequence types and serotypes. Virulome and resistome analyses revealed the preponderance of virulence genes and a reduced number of antimicrobial genes in Pks-positive isolates. This study strongly contributes to understanding the evolution of pks islands in E. coli.

BackgroundThe plant root microbiome is central to disease resistance and stress resilience. In intensive tomato production, prolonged agrochemical use disrupts microbial communities, reducing their protective functions and enabling pathogen establishment. MethodsWe integrated 16S rRNA amplicon sequencing with culture-dependent isolation to analyze microbiome shifts in tomato plants across healthy, asymptomatic, and symptomatic states in a nematode-infested field. Network analysis and machine learning were used to identify key taxa. Isolates were screened for plant growth-promoting rhizobacteria (PGPR) and nematicidal activity, and selected strains were evaluated in planta under pathogen challenge. ResultsMicrobial diversity and community complexity declined with disease severity. Gaiella occulta emerged as a potential biomarker of plant health. From 223 isolates, 45 strains exhibited PGPR and nematicidal traits. Ten were tested in tomato plants, where treatments conferred systemic resistance to Pseudomonas syringae pv tomato without fitness cost. Four strains, primarily Pseudomonas and Bacillus, triggered immune priming, enhanced root development, and three of them were co-isolated from a single asymptomatic plant. ConclusionsOur findings highlight the potential of targeted bacterial consortia to restore microbiome balance and activate immune responses in tomato. These results support the rational design of synthetic microbial communities (SynComs) for sustainable, microbiome-based crop protection.

Plant-associated microbiota affect pant growth and development by regulating plant hormones homeostasis. Indole-3-acetic acid (IAA), a well-known plant hormone, can be produced by various plant-associated bacteria. However, the prevalence of microbes with the capacity to degrade IAA in the rhizosphere has not been systematically studied. In this study, we analyzed the IAA degradation capabilities of bacterial isolates from the roots of Arabidopsis and rice. Using genomics analysis and in vitro assays, we found that 21 out of 189 taxonomically diverse bacterial isolates possess the ability to degrade IAA. Through comparative genomics and transcriptomic assays, we identified iac-like or iad-like operon in the genomes of these IAA degraders. Additionally, the regulator of the operon was found to be highly conserved among these strains through protein structure similarity analysis. Some of the IAA degraders could utilize IAA as their sole carbon and energy source. In planta, most of the IAA degrading strains mitigated Arabidopsis seedling root growth inhibition (RGI) triggered by exogenous IAA. Importantly, we observed increased colonization preference of IAA degraders from soil to root according to the frequency of the biomarker genes in metagenome-assembled genomes (MAGs) collected from different habitats, suggesting that there is a close association between IAA degraders and IAA-producers. In summary, our findings further the understanding of the functional diversity and roles of plant-associated microbes.

Vesicle transport is an essential process that mediates the growth, development and virulence of pathogenic fungi. However, the intricate mechanisms underlying how vesicle transport regulates the secretion of effector proteins remain to be fully elucidated. Here, we unveiled a novel pathway in which retromer and trans-Golgi (TGN) SNARE proteins co-regulate the proper secretion of apoplastic effectors in the rice blast fungus Magnaporthe oryzae. A TGN-associated SNARE complex consisting of MoSnc1, MoTlg1, MoTlg2, and MoVti1 was found to be essential for growth, development and pathogenicity in the fungus. Moreover, the TGN-associated SNARE complex is indispensable for accurate secretion of apoplastic effectors. Furthermore, we have elucidated that the dynamin-like protein MoVps1, an upstream regulator of the retromer complex, regulates the fission of MoVps35-coated vesicle and the proper localization of the TGN-associated SNARE complex. Additionally, we employed prochlorperazine, which identified as a potent dynamin inhibitor, elicits a developmental response in M. oryzae akin to MoVPS1 disruption, highlighting the pivotal regulatory role of dynamin and its potential as a therapeutic target for rice blast disease management. In conclusion, the study uncovered a specific mechanism by which MoVps1 and the retromer complex regulate the positioning of TGN-associated SNARE proteins to effectively promote effector secretion. It provides a deeper understanding of the molecular mechanisms of effector secretion in fungi and underscores the importance of vesicle transport in fungal pathogenesis. ImportanceVesicle transport is essential for pathogenic fungi as it controls the secretion of effectors that modulate interactions with the host and infection processes. The detailed mechanisms of effector secretion via vesicular pathways in these fungi are not yet fully understood. In this study, we have discovered a new regulatory pathway involving the retromer complex and trans-Golgi SNARE proteins that is critical for the proper secretion of apoplast effectors in M. oryzae. We have identified an important TGN-associated SNARE complex, consisting of MoSnc1, MoTlg1, MoTlg2 and MoVti1, which is required for the development and pathogenicity of M. oryzae. Our results emphasize the importance of this SNARE complex for the precise secretion of effectors into the apoplast, a key step in pathogenesis. Additionally, we demonstrated that the dynamin-like protein MoVps1, a protein acting upstream of the retromer complex, is vital for the correct localization of the TGN-associated SNARE complex. Furthermore, our research underscores the critical regulatory role of dynamin in M. oryzae pathogenesis, with prochlorperazine serving as an inhibitor that mimics the phenotypic effects of MoVps1 disruption, thereby highlighting its potential as a biopesticide candidate for rice blast disease management. Our study has uncovered a specific regulatory mechanism in which MoVps1 and the retromer complex control the positioning and function of TGN-associated SNARE proteins, thereby facilitating effector secretion. This work not only advances our understanding of the molecular basis of effector secretion in fungi, but also has implications for the development of novel strategies to control fungal diseases.