Molecular Plant-Microbe Interactions®

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.

Aphanomyces euteiches, the causative agent of Aphanomyces root rot (ARR), is of major concern for pea and other legume crops globally. This oomycete pathogen causes substantial decreases in crop yields, is unaffected by most fungicides, and persists in the soil for many years via its resilient oospores. Given the significance of pea crops in sustainable agriculture, namely the ability to fix nitrogen and act as a sustainable protein source, solutions to ARR are of high importance. We used RNA-seq in a novel strain of Pseudomonas donghuensis to identify two biosynthetic gene clusters under GacA/S control that are involved in producing bioactive molecules capable of inhibiting A. euteiches. Based on similarity to other reported clusters in Pseudomonas, the first is predicted to encode for a pseudoiodinine compound, while the second is predicted to produce the siderophore 7-hydroxytropolone. Individual knockouts of each cluster showed loss of inhibitory action of P. donghuensis NRC29 against A, euteiches in vivo. This is the first report highlighting the potential of P. donghuensis and the products of the two identified biosynthetic pathways as biocontrol agents for A. euteiches. Further investigations into the efficacy of P. donghuensis NRC29 and its metabolites in inhibiting A. euteiches in field trials will be of high value in developing sustainable strategies for ARR mitigation. ImportanceModern fungicidal treatments for control of root rot in pulse crops are ineffective for control of A. euteiches, leaving limited strategies for management of A. euteiches infected fields. We describe a novel P. donghuensis strain with potential for biocontrol against this persistent pathogen. Given the economic value of peas and other pulses globally, further work into harnessing the bioactive metabolites produced by this strain into a practical in-field treatment will be valuable.

Norway spruce (Picea abies) responds to attacks by the spruce bark beetle (Ips typographus) through the rapid activation of local defense mechanisms, but field studies can be difficult to standardize due to variable attack pressure and environmental heterogeneity. Here, we developed a phytotron-based assay that mimics early beetle-associated stress using insect-derived protein extracts, enabling reproducible molecular analyses under controlled conditions. Ten-week-old spruce seedlings were stem-treated with mock buffer or beetle protein extracts, followed by transcriptomic analyses of stem tissues and targeted metabolomic profiling of needles at 2 and 48 h post-inoculation. RT-qPCR analysis revealed rapid transcriptional activation of signaling and defense genes in Norway spruce, with NP-40-based protein extracts producing the most consistent early response. RNA-seq analysis revealed transcriptional dynamics, with 488 differentially expressed genes detected at 2 h and 84 at 48 h post-inoculation relative to mock-treated controls. Early responses at 2 h were characterized by activation of genes associated with immune perception and signal transduction. By 48 h, the response shifted toward accumulation of transcripts encoding defense proteins such as chitinases, defensins, proteinase inhibitors, and pathogenesis-related (PR) proteins. Importantly, a substantial proportion of differentially expressed genes overlapped with those previously identified in mature Norway spruce trees during pioneer bark beetle attack under field conditions, supporting the biological relevance of the assay. In contrast, targeted analyses of secondary metabolites performed in needle tissue revealed limited systemic changes across time points, suggesting that early induced defenses may remain largely localized to the stem. Together, these results demonstrate that beetle-derived proteins trigger a rapid and temporally structured defense response in Norway spruce seedlings and establish a reproducible elicitor-based platform for dissecting conifer immune responses and screening spruce genotypes for bark beetle resistance. HighlightBark beetle protein elicitors trigger temporally structured immune responses in Norway spruce seedlings that overlap with responses observed in mature trees, with rapid immune signaling at 2 h followed by defense protein accumulation at 48 h.

Colletotrichum siamense is a predominant causal agent of anthracnose in rubber tree and numerous economically important crops, causing severe yield losses worldwide. Conidial germination represents a critical early step for successful infection, while the high-osmolarity glycerol (HOG) MAPK pathway and ergosterol biosynthesis individually govern fungal development, stress adaptation and fungicide responses. However, the molecular crosstalk between these two modules remains largely elusive in phytopathogenic fungi. Here, we identified CsErg5B, a sterol C-22 desaturase homolog, as a direct target of the HOG- regulated transcription factor CsAtf1 in C. siamense. CsErg5B was indispensable for ergosterol biosynthesis, conidial germination, appressorium formation, and full virulence. The {Delta}CsErg5B mutant showed increased conidiation but severely impaired germination, and exhibited elevated resistance to fludioxonil while hypersensitivity to azole fungicides. Epistasis analysis using the {Delta}CsErg5B/{Delta}CsCyp51G1 double mutant - where CsCyp51G1 serves as another downstream target of CsAtf1 - revealed that CsErg5B functions as the predominant downstream effector of CsAtf1 in modulating conidial development and fludioxonil sensitivity. Furthermore, overexpression of CsErg5B significantly rescued the defects in conidial germination and fludioxonil sensitivity in both {Delta}CsAtf1 and {Delta}CsPbs2 mutants. Taken together, our findings uncover a HOG MAPK - CsAtf1 - CsErg5B regulatory axis that connects HOG MAPK signaling to ergosterol homeostasis, thereby governing conidial germination and fungicide sensitivity in C. siamense. This study provides novel insights into the regulatory network underlying fungal development and fungicide response, and offers promising molecular targets for the integrated management of plant anthracnose.

{gamma}-Glutamyl cyclotransferases (GGCTs) belongs to class of cytosolic enzymes that are responsible for glutathione (GSH) degradation under stress conditions. They regulate GSH homeostasis through the {gamma}-glutamyl cycle which is responsible for maintaining the synthesis of GSH as well as its breakdown, enabling recycling of its constituent amino acids. Although GGCTs have been implicated in enhancing heavy metal (HMs) tolerance in plants, their role in biotic stress remains largely unexplored. Previously, OsGGCT1 was identified as a gene strongly upregulated in Fusarium stress. In this study, the GGCT1 homolog from Oryza sativa japonica was characterized for its role in conferring tolerance to Fusarium oxysporum (F.O.). Similar to abiotic factors, biotic stresses significantly impact crop yield and productivity. The rhizosphere harbors diverse microbial communities, including harmful pathogens such as F. oxysporum. Fusarium causes wilt disease in a variety of plant species, such as: tomato, legumes, rice, and Arabidopsis thaliana. Our results demonstrate that overexpression of OsGGCT1 enhanced tolerance to F. oxysporum in A. thaliana, primarily by reducing fungal spore accumulation. Transgenic plants showed elevated expression of OsGGCT1 along with AtGSH1 and AtGSH2, reduced levels of reactive oxygen species (ROS), improved growth and photosynthetic performance and enhanced activities of the antioxidant enzymes. OsGGCT1 serves as a key component in maintaining GSH homeostasis by supporting glutamate (Glu) regeneration necessary for sustained GSH biosynthesis. Overall, these findings identify OsGGCT1 as an important constituent of the GSH-mediated detoxification pathway against Fusarium oxysporum and provide valuable molecular insights for developing Fusarium-tolerant rice varieties with reduced fungal accumulation.

Meloidogyne incognita is a major plant-parasitic nematode responsible for substantial yield losses in tomato worldwide. Current control strategies rely heavily on chemical nematicides, which raise environmental concerns and face increasing regulatory restrictions, underscoring the need for sustainable alternatives. Here, we show that foliar application of an aqueous extract from cavalcade (Centrosema pascuorum) enhances tomato resistance against M. incognita. Pre-inoculation treatment with cavalcade extract prior to inoculation with root-knot nematodes (RKN) significantly reduced root gall formation, delayed nematode development, and limited second-stage juvenile penetration compared with untreated infected controls, whereas post-inoculation application conferred partial protection. Transcriptomic analyses revealed the activation of multiple defense-related pathways, including salicylic acid- and jasmonic acid-associated signaling and phenylpropanoid metabolism, supported by the upregulation of PR1 and PAL. Additional induction of lipid transfer proteins, leucine-rich repeat receptor-like kinases, resistance proteins, mitochondrial calcium uniporter, and GA2ox5 suggests coordinated activation of pathogen recognition, calcium signaling, and hormone-regulated defense networks. These findings demonstrate that cavalcade extract primes broad-spectrum defense responses in tomato and highlight its potential as an environmentally sustainable strategy for nematode management.

O_LISymbiosis between legumes and rhizobia is beneficial on nutrient-poor soils, as it enables the fixation of atmospheric N2. To establish this symbiosis, gene expression in both the host plant and the symbiont has to be regulated. To understand the underlying RNA-mediated regulation of host gene expression, we designed experiments to identify competing endogenous networks involving circular RNA, microRNA, and linear transcripts during symbiosis, using wt and symbiosis-deficient Lotus japonicus mutants with the rhizobium Mesorhizobium loti (M. loti). C_LIO_LICircRNA, miRNA, and linear transcripts were identified from Lotus japonicus wildtype and CCamK mutant (ccamk-13; snf-1) seedlings without inoculation or with M. loti inoculation using deep short-read sequencing with rRNA-depletion and random primers. C_LIO_LIDifferentially expressed miRNAs showed negative correlations to predicted target genes and may regulate symbiotic processes. The symbiosis essential iron-sensor LjnsRING/BRUTUS expresses a circRNA which was upregulated in symbiotic treatments. This circRNA may act as a target mimic and contribute to nodule longevity. CircRNAs are predicted to act predominantly as trans-regulatory molecules with similar frequencies in Arabidopsis thaliania, Oryza sativa, and Lotus japonicus. C_LIO_LIWe identified novel miRNAs, long noncoding RNAs, and circRNAs, and nominated several as potential new regulatory non-coding RNAs that may act as target mimics to stabilize genes and support symbiosis. C_LI SummarySymbiosis between Lotus japonicus and Mesorhizobium loti involves treatment-specific regulation of competing endogenous RNA networks involving circular RNA, miRNA, and linear transcripts.

Glutathione (GSH) is a tripeptide that plays essential roles in redox regulation and stress responses across organisms. In Escherichia coli, the GSH-specific {gamma}-glutamyl cyclotransferase (ChaC) has been characterized biochemically, yet its physiological role remains unclear. Moreover, ChaC has been annotated as a regulator of the Na/H antiporter ChaA based on its genomic association, although experimental evidence supporting this function is limited. In this study, we investigated whether chaC and its co-transcribed gene, chaB, are involved in sodium transport or GSH metabolism. Gene expression analyses revealed that chaA, chaB, and chaC are upregulated under salt stress. Functional analyses using deletion mutants showed that loss of chaA reduced salt tolerance, whereas deletion of chaB enhanced tolerance and decreased intracellular sodium levels. In contrast, deletion of chaC had no significant effect on salt tolerance or sodium accumulation. Overexpression of cha genes further indicated that chaA, but not chaB or chaC, contributed to salt tolerance. Importantly, overexpression of chaC significantly reduced intracellular GSH levels, whereas chaB overexpression had no effect. These results indicate that ChaC primarily functions in GSH degradation rather than in cation transport, and that ChaB does not participate in GSH metabolism. Our findings clarify the distinct physiological roles of ChaC and ChaB and provide new insight into bacterial physiology regarding GSH metabolism and ion transport in E. coli.

Plant immunity can be activated by membrane-localized pattern recognition receptors or by cytosolic nucleotide-binding leucine-rich repeat (NLR) receptors, while it can be counteracted by pathogen secreted effectors. Manifestation of immunity often involves endomembrane traffic. However, limited evidence is available for such an involvement in NLR-mediated immunity, which typically includes a hypersensitive reaction (HR)-programmed cell death response. Here we show that the barley powdery mildew fungus uses several effectors to target and inhibit the vacuolar trafficking pathway, which causes endomembrane markers to stall in the endoplasmic reticulum (ER). We used this ER-stalling phenotype as a proxy to track fungal manipulation of the vacuolar pathway during different stages of the actual infection. Our data indicate that powdery mildew fungi interfere with the vacuolar pathway, but only temporarily, as the stalling is lifted once the fungal haustorial feeding structures are well-developed for nutrient uptake in epidermal cells. Notably, we show evidence that blocking the vacuolar pathway causes a general inhibition of NLR-mediated HR, and that this mechanism is taken advantage of by the pathogen. Finally, we provide an example that the fungus can secrete a different set of effectors at the haustorial stage that result in the re-opening of the vacuolar pathway by inhibiting the initial vacuolar traffic-suppressing effectors.

Pattern recognition receptors (PRRs) mediate plant immune responses by detecting extracellular immunogenic patterns, including microbe-associated molecular patterns (MAMPs). PRR signaling is commonly assessed using assays such as reactive oxygen species (ROS) bursts, cytosolic calcium influx, mitogen-activated protein kinase (MAPK) activation, and seedling growth inhibition (SGI), which are performed in distinct experimental systems, including seedlings grown on artificial media and soil-grown rosettes. Here, we systematically compare receptor kinase immune outputs triggered by the bacterial MAMPs elf18 and flg22 in Arabidopsis thaliana seedlings and rosettes across a range of concentrations. Rosettes exhibited greater sensitivity than seedlings in ROS assays, whereas cytosolic calcium responses measured using the Aeqcyt/pMAQ2 reporter were stronger in seedlings, correlating with reduced reporter transcript accumulation in rosette tissue. MAPK activation was consistently stronger in rosettes, whereas SGI assays revealed higher sensitivity to elf18 than flg22 in seedlings despite flg22 inducing stronger early signaling outputs. Together, these results demonstrate that canonical PRR-mediated immune outputs are differentially sensitive to experimental context and should not be interpreted as interchangeable measures of immune activation. These findings highlight the importance of considering experimental conditions when comparing immune responses across assays and developmental stages.

O_LIHost specialization is a major driver of genetic structure in fungal plant pathogens, but it remains unclear whether specialization on different cereal hosts prevents sexual recombination when mating-type compatibility is retained. We addressed this question in stripe rust, caused by Puccinia striiformis, by crossing wheat-adapted P. striiformis f. sp. tritici and barley-adapted P. striiformis f. sp. hordei, two divergent host-adapted forms that share common barberry (Berberis vulgaris) as a sexual host. C_LIO_LIControlled reciprocal crosses on barberry produced 18 aeciospore-derived progeny, demonstrating that wheat- and barley-adapted Puccinia striiformis can undergo sexual recombination despite strong host specialization during asexual infection. Chromosome-scale parental assemblies placed the homeodomain (HD) mating-type locus, containing bW-HD1 and bE-HD2, on chromosome 2 and the pheromone receptor (PR) mating-type locus, containing STE3 and mfa genes, on chromosome 6. HD restriction genotyping showed biparental inheritance in all progeny, with each progeny carrying one HD haplotype from each parent. Together with conservation of PR-associated coding sequences and amplification of STE3-associated markers in progeny, these results are consistent with retention of tetrapolar mating across the two host-adapted lineages. C_LIO_LIHost interaction phenotypes were assessed across wheat and barley differentials, near-isogenic lines and wild relatives. The parental isolates retained contrasting wheat- and barley-restricted profiles, whereas progeny did not reproduce either parental virulence profile, but instead showed recombinant infection patterns, including compatibility with both wheat and barley genotypes. C_LIO_LIThese findings indicate that host specialization in Puccinia striiformis does not necessarily prevent sexual compatibility on a shared alternate host. Together with retention of tetrapolar mating, alternate-host sexual reproduction may provide a route for genetic exchange between host-specialized pathogen populations, enabling recombination to generate new combinations of host-interaction traits when divergent pathogen lineages mate on a shared alternate host. C_LI

Magnaporthe oryzae, the rice blast fungus, plays a role as a model organism for molecular plant-microbe interaction research. Studies on the pathogenic mechanism of this fungus revealed many genes involved in signaling pathways. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological processes during the pathogenesis of M. oryzae. Identifying the genome-wide protein-protein interaction (PPI) network is one of the omics-level approaches, which helps to understand signaling and regulatory pathways. However, existing biological network resources of M. oryzae are not sufficient to decipher pathogenesis mechanisms due to the abundance of false positives/negatives. In this study, a reliable PPI network database of M. oryzae, MagNet, was constructed with three methods, including homology-based Interolog search, co-expression network construction, and domain-domain interaction (DDI)-based prediction. With three approaches altogether, the pan-network with 5,600,976 interactions was generated, including 217,531 highly confident interactions supported by all three methods. Experimental data on M. oryzae PPIs supported that our PPI network can predict PPIs with higher accuracy compared to the previously constructed databases. MagNet would provide integrated biological network data, which can help to understand the molecular mechanisms of the rice blast fungus. The PPI data can be accessed via https:/magnet.scnu.ac.kr.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) of Gram-negative pathogenic bacteria recognized by plants, triggering typical pattern-triggered immunity (PTI) responses. However, a LPS sensing receptor for the recognition of plants remains largely undefined. A plant receptor for lipopolysaccharide (LPS) has not yet been identified. Here, we identify a plant protein, OsML1, with homologies to animal MD-2, which is capable of binding LPS. Furthermore, it may act as a molecular chaperone to assist CK1 in perceiving LPS signals. Our results show that OsML1 functions as an LPS-binding protein recognizing LPS and participates in downstream rice immune response activation. Structural modeling and sequence analysis revealed that OsML1 contains both a typical ML domain and a conserved three-dimensional {beta}-barrel structure as mammalian MD-2 proteins. Microscale thermophoresis assays confirmed that OsML1 binds LPS with high affinity. Functional analyses further demonstrated that OsML1 knockout plants show reduced resistance to the rice bacterial blight pathogen, as well as attenuated ROS bursts upon LPS treatments, whereas overexpression plants show enhanced immune responses. Metabolomic profiling indicated significant metabolic changes in OsML1 knockout plants, particularly in immune-related pathways involving lipids, amino acids, and antimicrobial compounds. OsML1 is consequently a structurally conserved and functional LPS-binding protein linking lipid metabolism, LPS perception, immune activation, and metabolic regulation. Phylogenetic and structural analyses revealed that OsML1 likely arose from a duplication of OsML2, forming an independently functional subgroup within the PITP family. Our study identifies OsML1 as a LPS recognition factor involved in LPS sensing and downstream ROS bursts activation, callose deposition, and broad-spectrum gene expression of resistance. These findings expand our knowledge of bacterial LPS perception and immune regulation in plants, offering novel targets and strategies for disease-resistant breeding.

The root cap is essential for perceiving environmental cues surrounding the root. However, the molecular mechanisms underlying root cap-mediated immunity and how it defends against invading pathogens remain largely unresolved. Our results indicate that cytokinin plays a major role in regulating soil-borne pathogen such as Ralstonia pseudosolanacearum load around the root and root cap. As Ralstonia populations increase, cytokinin signalling is activated and represses the expression of its downstream signalling targets such as root cap-specific proteins JAL10 and JAL20, to impart the tolerance against the Ralstonia. The functional analysis jacalin-associated lectin family proteins JAL10 and JAL20, revealed that loss-of-function leads to enhance tolerance to Ralstonia whereas gain-of-function leads to susceptibility compared to Col-0. Our Glycoproteomic and metabolomic analyses indicate that JAL10 and JAL20 act as negative regulators of cell wall remodelling and likely to promotes cell wall thickening, thereby enhancing resistance to soil-borne infections. The knockdown of ortholog of JAL protein in Tomato also revealed its conserved function in imparting tolerance to Ralstonia pseudosolanacearum. Further we also show downregulation of JALs by other soil-borne pathogen infection, suggesting that cytokinin might protecting the vulnerable areas of root tip regions by regulating the expression of root cap-specific JALs and thereby fortifying the cell wall.

Bacterial spot is a consistent threat to global tomato and pepper productions; however, Ohios fresh market production currently lacks the updated surveillance data necessary to provide accurate management solutions. While traditional diagnostics focus on identification of a single causal agent, shotgun metagenomic sequencing (MGS) offers a comprehensive view of the infection court. An assignment-first MGS workflow was developed and validated in this study, utilizing Kraken2 databases to extract Xanthomonas species associated with bacterial spot and to characterize the microbial communities of bacterial spot in Ohio production systems. Through in silico spiking experiments, thresholds were established for bacterial spot identification. Species and pathovar identification via average nucleotide identity (ANI) remained accurate at abundance as low as 0.1%. A minimum of 2% Xanthomonas reads were required for high genome completeness (BUSCO >90%) and 3% for reliable type III secretion system (T3SS) effector profiling. Analysis of 63 samples from fresh-market production fields identified Xanthomonas hortorum pv. gardneri, Xanthomonas euvesicatoria pv. euvesicatoria, and Xanthomonas arboricola residing in symptomatic samples, alongside other taxa including Pseudomonas and Stenotrophomonas. Phylogenetic comparisons of metagenome-assembled genomes (MAGs) were comparable to whole genome sequences (WGS) from the same samples, supporting the reliability of culture-independent diagnostics. These results provide a robust framework for utilizing metagenomics as a diagnostic tool, expanding our knowledge of bacterial spot population structure in Ohio, and uncovering the bacterial communities associated with bacterial spot.

The persistence of specialised survival spores produced by microbial pathogens represents a primary bottleneck in the management of plant diseases. In oomycetes, these spores (known as oospores) are largely impervious to chemical control, allowing them to persist in soil and initiate new infection cycles over many years. A prominent example is the soil-borne pathogen Phytophthora agathidicida, the causal agent of kauri dieback disease, where long-lived oospores hinder conservation efforts in native forests. The resilience of oospores is attributed to their thick wall composed of complex {beta}-glucan layers that render the oospores impermeable to most conventional biocides. Here we have investigated an enzyme-based approach for weakening the oospore cell wall. We searched enzyme databases to select {beta}-glucanases targeting a variety of linkages found in Phytophthora oospore walls. Eight of these {beta}-glucanases were successfully purified and tested for their digestive activity against intact oospores in vitro using a phenol-sulfuric acid assay. We showed that combining these enzymes was crucial to achieve significant digestion through synergies and additive effects. The optimal combination, comprising 1,3-, 1,6-, and 1,3(4)-{beta}-glucanases, was evaluated for its ability to permeabilise oospores to five biocides typically effective only on other, more sensitive lifecycle stages of the pathogen. Using a live/dead fluorescence assay, we observed that the effects of the membrane-targeting biocides were potentiated in oospores that were pre-treated with the {beta}-glucanase mixture. Our results highlight enzymatic cell wall permeabilisation as a promising strategy toward improved management of oospore persistence in kauri forest soils and against broader oomycete threats. KeypointsO_LIOur phenol-sulfuric acid assay can be used to screen for oospore-degrading enzymes. C_LIO_LISynergistic enzyme combinations are essential for effective oospore wall digestion. C_LIO_LIEnzyme pre-treatment sensitises oospores to membrane-targeting biocides. C_LI

Some filamentous plant-pathogenic fungi have comparably large genome sizes within the fungal kingdom due to the proliferation of transposable elements (TEs). Blumeria hordei (Bh), the causal agent of the powdery mildew disease on barley, is a filamentous obligate biotrophic fungus. Compared to other ascomycetes, it contains a low number of genes but a high genomic TE content of approximately 75%. Yet, a comprehensive understanding of the contribution of TEs to the RNA and protein landscape of Bh is lacking. Here, we use Bh as a model to study transcripts and proteins derived from genes and individual TEs. Therefore, we created two high-quality genome assemblies of the German Bh isolate TUM1 and the Australian Bh isolate AUS1. We applied deep proteomics with mass spectrometry, long-read and short-read sequencing on both DNA and RNA. Based on these multi-omic resources, we completed nearly gapless genome assemblies, new gene and TE annotations, and effector predictions. Using long-read RNA sequencing, we detected extensive co-transcription of TEs and genes as TE-gene chimeric transcripts. We identified previously unpredicted splice variants or genes, partially supported by proteomics. The intergenic and TE genomic space of Bh TUM1 gives rise to thousands of transcripts and several novel TE-derived proteins that lack from previous TE protein predictions. Together, this supports an existing potential for expression of novel transcripts and proteins from highly abundant TEs in the Bh genome.

Insect herbivory triggers cytosolic proteome reprogramming by activating defense pathways and modulating key metabolic processes. We found that simulated herbivory in pigeon pea (Cajanus cajan) induced reactive oxygen species (ROS) production and molecular alterations within 12 hours (h) of post treatment. We compared the leaf proteome profiles of two cultivated genotypes, ICPL 332 (moderately resistant) and ICPL 87 (susceptible), using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry (MS). More than 220 protein spots were detected in ICPL 332 and over 200 in ICPL 87. Comparative analysis revealed 75 differentially accumulated proteins (DAPs), of which 40 were consistently reproducible across biological replicates. These included 11 unique to ICPL 87, 9 unique to ICPL 332, and 10 common to both genotypes. Among the shared DAPs, ICPL 332 showed five upregulated and five downregulated, whereas ICPL 87 exhibited only two upregulated and eight downregulated. Functional categorization grouped DAPs into primary metabolism, stress response, and growth and development. Proteins related to primary metabolism were largely downregulated in both genotypes, while stress-associated proteins exhibited substantial downregulation in ICPL 87 compared to ICPL 332. Overall, the results demonstrate proteomic adjustments underlying defense responses in pigeon pea genotypes.

Functional validation of genetic components in plants often requires cloning them separately into both plant and bacterial expression vectors, a process that is both time-consuming and laborious. This study aimed to simplify this workflow by developing plant-bacteria dual-host promoter systems that drive high-level constitutive expression in both environments. To achieve this, two variants of the chloramphenicol acetyltransferase promoter (PCAT), a bacterial {sigma} factor-dependent promoter, were integrated into the cauliflower mosaic virus 35S promoter (P35S), and their performance was evaluated using a hygromycin phosphotransferase (HPT)-GFP fusion reporter. One of these variants, PCAT1, conferred hygromycin resistance to Escherichia coli (DH5 and BL21 (DE3)) and maintained high-level expression comparable to the original P35S in onion epidermal cells. A hybrid P35S enhancer-PNOS system also conferred hygromycin resistance to E. coli, but its activity in inducing GFP signals in onion cells remained lower than that of P35S. Due to its compact size (89 bp) and efficiency, PCAT1 can serve as a module for converting standard plant vectors into dual-host systems, accelerating gene characterization and the development of new gene-based tools.

Antibiotic use in agricultural systems can unintentionally disrupt beneficial rhizosphere microorganisms, yet the consequences of this dysbiosis for plant fitness remain insufficiently understood. Building on previous findings that application of streptomycin to the roots decreases cyanobacteria and increases tomato plant susceptibility to foliar Xanthomonas infection, this study aimed to determine whether this relationship reflects causation or correlation. We evaluated whether targeted inoculation with the filamentous nitrogen-fixing cyanobacterium Cylindrospermum sp. (CI) or a complex rhizosphere microbiome transplant (RMT) could mitigate antibiotic-induced dysbiosis. As expected, streptomycin treatment significantly increased bacterial spot disease severity and reduced microbial richness in the rhizosphere, marked by a pronounced decline in cyanobacterial and Cylindrospermum operational taxonomic units. Co-occurrence network analysis revealed that this dysbiotic state was defined by reduced community connectivity and increased negative associations, indicating a breakdown in cooperative microbial relationships. Notably, both CI and RMT reduced plant disease severity, though they caused distinct rhizosphere community reassembly outcomes. While RMT relied on microbial functional redundancy, the targeted CI approach achieved more robust colonization and effectively "patched" the functional gap left by dysbiosis. Microbiome restoration directly influenced host physiology, significantly reducing the overactivation of ethylene-mediated defense genes, such as ERF1, and partially reinstating auxin-responsive signaling pathways (IAA21) that were disrupted under dysbiosis. These findings suggest that targeted microbial inoculation could reverse dysbiosis and enhance plant resilience under pathogen pressure as effectively as complex microbial transplants. This work highlights a shift in microbiome management: from the complex rebuilding of communities to the strategic repair of specific functional gaps.