Molecular Plant-Microbe Interactions®

Pyrenophora teres f. teres (Ptt), the causal agent of net form net blotch disease in barley, is an economically important fungal pathogen worldwide. Understanding both host resistance mechanisms and pathogen virulence factors is essential for developing durable net form net blotch resistant barley cultivars. Quantitative trait loci (QTL) mapping was conducted using a cross between two Ptt isolates, one virulent on the barley cultivar Prior and the other being avirulent. A major QTL associated with virulence on Prior was detected on chromosome 5. A progeny isolate possessing this QTL, together with the two parental isolates, was subsequently used in the proteomic analyses. Label-free proteomics was used to quantify in planta the protein profile changes in Prior following inoculations with the virulent and avirulent parental Ptt isolates, and the virulent progeny isolate. Leaf samples were collected at two (D2) and five (D5) days post-inoculation, and proteomic analyses performed to identify proteins associated with host resistance and pathogen virulence. A dataset comprising 2,886 barley proteins and 51 Ptt proteins was analysed. Principal component analysis (PCA) of the barley Prior proteomes revealed distinct clustering based on resistance and susceptibility at D5, while D2 samples formed a separate cluster. The PCA of the Ptt proteomes identified separate clusters, one comprised of the D2 and D5 avirulent parental isolate and another cluster of the virulent isolates at D5 only. Gene ontology analysis of the Prior proteins that were significantly increased in the resistant compared to the susceptible groups revealed functional categories related to protein translation, biosynthesis and chloroplast activities. The proteins that were significantly increased in the susceptible compared to the resistant Prior group were associated with organic acid and carbohydrate metabolism. The proteomic profiles and bioinformatic analysis generated in our study provide novel insights into the molecular basis of resistance and virulence in the barley-P. teres pathosystem. Key messageThis study reveals the first in planta proteomic profiles of both barley and Pyrenophora teres f. teres, identifying unique virulence-associated proteins and host responses linked to resistance and susceptibility.

Pyrenophora teres f. teres (Ptt), the causal agent of net-form net blotch in barley, was studied using a bi-parental mapping population (Pop1) of 305 isolates derived from a cross between two isolates with contrasting virulence on barley cultivars Skiff and Prior. QTL analysis identified virulence loci on chromosomes (Chr) 3 and 10 for Skiff, and on Chr 1, 4, and 5 for Prior. Major QTL on Chr 3 and 5 explained 24% and 40% of phenotypic variation, respectively. A second population (Pop2) was developed by crossing two Pop1 isolates, one carrying major QTL on Chr 3 and 5 and one avirulent. Isolates from Pop2 with single QTL were phenotyped across a Prior/Skiff recombinant inbred line population to identify corresponding host susceptibility/resistance loci. Skiff virulence QTL on Chr 3 corresponded to barley Chr 3H and 6H, while Prior virulence QTL on Chr 5 mapped to Chr 6H. RNA expression analysis of virulent and avirulent Pop2 isolates identified five candidate genes linked to the Chr 5 QTL, including two predicted effectors. These findings suggest both gene-for-gene and inverse gene-for-gene interactions in the Ptt-barley pathosystem and advance the understanding of molecular mechanisms underlying host-pathogen specificity.

Fungal pathogens are responsible for substantial crop losses worldwide. There is a pressing need to develop crops with improved disease resistance, especially given that climate change and human activities are exacerbating crop diseases. Our understanding of the molecular mechanisms by which fungi cause disease is incomplete. To address this limitation, we employed proteomics to identify candidate effector proteins from the pathogenic fungus Ustilago maydis that co-purified with the chloroplasts of maize host plants during infection. We specifically characterized the role of one putative chloroplast-associated effector, UmPce3, using heterologous expression in the non-host plant Arabidopsis thaliana. We discovered that UmPce3 interacts with the chloroplast DEAD-box RNA helicase, AtRH3. Phenotypes associated with the expression of UmPce3 in Arabidopsis mirrored those of plants with impaired AtRH3 function and included interference with chloroplast assembly, an impact on photosynthesis, and altered resistance to biotic and abiotic stresses. Support for RH3 as a bona fide effector target was obtained by identifying parallel phenotypic influences of UmPce3 in maize and by demonstrating an interaction between UmPce3 and maize ZmRH3b, an ortholog of AtRh3. Notably, UmPce3 contributes to biotrophy by promoting the virulence of U. maydis on maize seedlings and dampening virulence in plants challenged with salinity as an abiotic stress. Overall, this work highlights the chloroplast as a target of fungal pathogenesis and identifies RH3 as a potential hub for pathogen manipulation of organelle function to balance fungal proliferation and host health in support of biotrophy. Short summaryThe chloroplast plays a key role in plant immunity, in addition to its central contributions to photosynthesis, metabolism, and tolerance of abiotic stresses. The effector UmPce3 of the maize pathogen Ustilago maydis targets the DEAD-box RNA helicase RH3 in host plants to manipulate chloroplast function and enhance fungal pathogenesis. Unexpectedly, UmPce3 also influences host tolerance to salt stress thereby balancing the plant response to biotic and abiotic stressors in support of biotrophic development.

During host-pathogen interactions, fungal pathogens secrete effector proteins into host cells to manipulate the host immune system and facilitate infection. Although many effector genes are highly expressed during infection stages, there is limited information on the mechanisms regulating their in planta expression. Here, we characterize the promoter of MoHTR1, a nuclear effector gene of the rice blast fungal pathogen, to elucidate its in planta-specific expression. Using promoter deletion and mutation analyses, we identified a core cis-element (TATTTCGT) within the MoHTR1 promoter, designated the in planta active (IPA) element, which is crucial for in planta-specific expression. The IPA element is responsible for the expression of not only MoHTR1, but also other effector genes including a known effector Slp1. Furthermore, the IPA element enables the in planta expression of MobZIP14, a gene specifically expressed during vegetative growth. The IPA element plays a critical role in fungal virulence by enabling MoHTR1 expression and regulating host immune responses. Bioinformatic and DNA-protein interaction analyses revealed that RGS1, a transcription factor containing a winged-helix binding domain, acts as a transcriptional regulator of MoHTR1 by directly binding to the IPA element. Our findings provide new insights into the regulatory mechanisms driving the in planta-specific expression of fungal effector genes.

Generalist pathogens infect diverse plant hosts, yet how these interactions differ across hosts is poorly understood. Here, we conduct a molecular analysis of a generalist pathogen interacting with closely related hosts. A co-transcriptomic framework is used to dissect host-pathogen interactions between the generalist necrotroph Botrytis cinerea and two closely related legume hosts, common bean (Phaseolus vulgaris) and cowpea (Vigna unguiculata). Using a diverse set of 72 Botrytis isolates, we quantified lesion development alongside host and pathogen gene expression. Although lesion formation was driven primarily by pathogen genetic variation, transcriptomic responses in both host and pathogen exhibited significant host x isolate interactions. This indicated that extensive, fine-scale transcriptional plasticity created similar disease outcomes. Botrytis genes showing host-specific expression were enriched for cell wall-modifying enzymes and some specialized metabolic genes, indicating greater host responsiveness of these core virulence mechanisms than previously appreciated. Co-expression network analysis in both host and pathogen further showed that in both organisms, gene membership for individual networks are restructured in response to genetic diversity. For example in Botrytis, we identify different sets of genes host-dependently co-expressing with a non-ribosomal peptide synthetase (NRPS) gene cluster, suggesting divergent functional deployment of the same virulence machinery across closely related hosts. Both legume species exhibited extensive isolate-dependent transcriptional reprogramming, with approximately two-thirds of expressed host genes responding to pathogen diversity. While conserved defense pathways such as jasmonate/ethylene signaling and phenylpropanoid metabolism were upregulated in both hosts, the specific genes in the networks differed markedly, highlighting lineage-specific rewiring of defense strategies. These results suggest that generalist pathogen success is underpinned by pervasive gene expression plasticity in both host and pathogen, allowing similar phenotypic outcomes to emerge from highly divergent molecular states. SummaryO_LIGeneralist pathogens infect diverse plant hosts, yet how these interactions differ across hosts is poorly understood. This study investigates how a generalist pathogen achieves successful infection across closely related hosts, and how these hosts respond. C_LIO_LIA co-transcriptomic approach was applied to interactions between 72 genetically diverse isolates of the fungal necrotroph Botrytis cinerea and two legume hosts, common bean and cowpea. Lesion development and host and pathogen gene expression were quantified. C_LIO_LILesion formation was primarily driven by pathogen genetic variation, yet both host and pathogen transcriptomes showed strong host x isolate interactions. Both host and pathogen balance conserved responses with finely tuned, host-specific mechanisms. Further, host-dependent transcriptional responses involve network modulation around a common core of genes in both host and pathogen. C_LIO_LIGeneralist pathogen success is underpinned by pervasive gene expression plasticity in both host and pathogen, allowing similar phenotypic outcomes to emerge from highly divergent molecular states. C_LI

Plants employ cell surface receptors to recognize pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity, a crucial defense mechanism against invading pathogens. Pep-13 is a PAMP derived from a class of conserved cell wall transglutaminases present in Phytophthora species, and its receptor PERU was reported recently. In our parallel study, we observed distinct responses to Pep-13 between two diploid potato inbred lines: E454 recognizes Pep-13, whereas A018 does not. Genetic analysis demonstrated that Pep-13 recognition in E454 is controlled by a single genetic locus, tentatively designated TGER (Transglutaminase elicitor response). Through bulked segregant analysis sequencing, followed by complementation assays, we found that the TGERa gene in E454 is essential for Pep-13 recognition. Sequence alignment revealed that TGERa shares 99.91% amino acid sequence identity with PERU, indicating that TGERa and PERU are allelic variants of the same gene (PERU/TGERa). TGERb, a highly homologous gene of TGERa, was identified in the E454 genome; notably, TGERa, but not TGERb, can recognize Pep-13. We further demonstrated that TGERb exhibits defects in both ligand binding and association with the co-receptor StSERK3A. Additionally, we found that the TGERa allele in A018 is a weak allele with reduced expression levels, presumably resulting from a 3 kb DNA fragment insertion in its first intron. Heterologous introduction of TGERa into Nicotiana benthamiana and tomato significantly enhanced their resistance to Phytophthora infestans. Collectively, our findings confirm that PERU/TGERa functions as the Pep-13 receptor in potato and provide a valuable molecular target for improving Phytophthora resistance in plants.

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.

O_LISalicylic acid (SA) and auxin are key regulators of plant immunity and development. The clubroot pathogen Plasmodiophora brassicae encodes PbGH3, an effector related to the GH3 family involved in phytohormone homeostasis. Although PbGH3 was proposed to conjugate auxin in vitro, its biological function in planta has remained unclear. This study aimed to determine the in vivo role of PbGH3 during host colonization. C_LIO_LIWe generated Arabidopsis thaliana and Brassica napus lines overexpressing PbGH3 and characterized their developmental phenotypes, hormone profiles, gene expression, and susceptibility to infection. Structural modeling was performed to assess PbGH3 similarity to plant GH3 proteins, and functional complementation was tested using the Arabidopsis gh3.12 mutant. C_LIO_LIThe expression of PbGH3 in Arabidopsis induced auxin-related developmental phenotypes without detectable accumulation of auxin conjugates. Instead, PbGH3 structurally and functionally resembled GH3.12/PBS3 inducing increased conjugated SA levels, reduced jasmonic acid, suppressed PIN2 expression, and increased root hair number and infection. PbGH3 complemented SA-related defects in the gh3.12 mutant. C_LIO_LIPbGH3 functions as a modulator of SA metabolism rather than an auxin-conjugating enzyme, likely competing with host GH3.12/PBS3 to constrain effective SA accumulation. This reveals a novel strategy by which P. brassicae disrupts SA-auxin homeostasis to promote host colonization and ensure disease development. C_LI PLAIN LANGUAGE SUMMARYThis study shows that the clubroot pathogen uses a protein called PbGH3 to modify the plants salicylic acid balance. This alters root traits and increases susceptibility to infection. Arabidopsis and canola plants engineered to produce PbGH3 showed similar changes, revealing that the pathogen uses this protein to disrupt hormone regulation and create conditions that support its colonization.

Crops face attacks from a wide range of pathogens and pests that deploy various parasitic strategies. All aggressors must manipulate major plant functions, such as immune signaling, hormone pathways, metabolism, and development, to successfully establish themselves and complete their life cycle. Here, we compared the tomato plants response to three evolutionary distant pathogens (nematodes, aphids and oomycetes) during compatible interactions using a transcriptomic approach. We identified differentially expressed genes and biological processes, and highlighted potential key host hubs associated with successful parasitism. By integrating recent published datasets, we refined our understanding of the global and tissue-specific mechanisms targeted during compatible interactions and, through co-expression analysis, identified clusters showing shared dysregulation patterns enriched in specific GO terms. Finally, model-to-crop translational analysis using the Arabidopsis interactome network, repositioned tomato candidates within larger interaction networks and emphasized the key positions occupied by some of them. Identifying these pivotal tomato targets is crucial to decipher processes underlying parasitism and, consequently, offers new opportunities to develop sustainable multi-pathogen control strategies.

Botrytis cinerea is a necrotrophic fungal pathogen that infects thousands of plant species. During infection, these diverse plant hosts produce different specialized metabolites that can inhibit pathogen growth and shape pathogen fitness. However, the genetic architecture of pathogen resistance toward individual host defense metabolites remains poorly understood. To address this question, we exposed 83 B. cinerea isolates to the metabolite linalool and quantified metabolic and structural responses. Exposure revealed extensive phenotypic diversity across isolates. Genome-wide association identified 101 genes of interest associated with membrane transport and stress response regulation. Genetic associations were stronger for morphological traits than for metabolic traits, suggesting that hyphal architecture may have a complex genetic architecture contributing to linalool resistance. Together, these results establish natural variation in linalool response and provide candidate loci for understanding how generalist pathogens respond to host-derived chemical defenses. Article SummaryTo understand how a generalist pathogen responds to host defenses, we asked how Botrytis cinerea responds to linalool, a widespread monoterpene involved in plant defense. We exposed 83 B. cinerea isolates to 1000 {micro}M of linalool for 72 hours and quantified metabolic traits (growth curves and growth dynamics over time) and morphological traits (hyphal network features). Using GWA, we linked phenotypic variation to genetic variants. Results indicate substantial natural variation in linalool resistance and distinct genetic architectures across trait classes: metabolic responses are driven by a relatively small number of loci with larger effects, whereas structural/morphological responses appear more polygenic.

Host resistance is a critical component of oat crown rust disease management. Pc94 is a qualitative resistance locus derived from diploid Avena strigosa with several independent introgressions into A. sativa that have been used in cultivar deployment. Quantitative trait locus (QTL) analysis combining previously published data for a historic A. strigosa population segregating for Pc94 revealed a large effect QTL on the distal end of A. strigosa chromosome 7A. Genome assembly of the parents identified a cluster of five nucleotide binding site leucine-rich repeat receptor (NLR) candidate genes within the QTL region. A single candidate NLR with an integrated zinc finger BED domain, AstNLR94, was determined as necessary for Pc94 resistance based on map-based cloning and forward mutagenesis. A presence/absence allele specific PCR marker was designed in AstNLR94 and verified for accuracy and specificity in a diverse panel of A. strigosa and A. sativa. Pc94 introgressions in A. sativa ranged in size from 1.7-71 Mbp and two different introgression locations appear to have occurred. In A. sativa Leggett, a 6.3 Mbp Pc94 introgression is located at the end of chromosome 2A, and the same sized introgression was discovered in the OT3098 v2 genome. Finally, a QTL analysis identified an additional minor resistance locus on A. strigosa chromosome 4A, which has complicated previous efforts to characterize the Pc94 locus. This is the first report of an NLR gene underlying disease resistance in Avena spp. and delivers a Pc94 marker for marker assisted selection to produce disease resistant cultivars. Key messageWe mapped a zfBED-NLR encoding gene necessary for Pc94 resistance, developed a diagnostic marker, and revealed diverse introgression sizes, clarifying Pc94s history and utility for durable oat crown rust resistance.

Plant immunity relies on the detection of microbes and the rapid activation of intracellular defense pathways. Catalyzed by protein kinases and E3 ubiquitin ligases, respectively, phosphorylation and ubiquitination are among the most abundant post-translational modifications that regulate immune pathways. It has been well established that members of the receptor-like cytoplasmic kinase (RLCK) and plant U-box E3 ligase (PUB) families are critical components of plant immune signaling. Interestingly, a group of proteins that contain both an RLCK domain and a PUB domain has been conserved throughout plant evolution, referred to as subgroups RLCK-IXb and PUB-VI within their respective families. While very little is known about these proteins, evidence from multiple independent studies indicates that orthologous PUB-VI/RLCK-IXb proteins in potato, tomato, Nicotiana benthamiana, and Arabidopsis thaliana associate with diverse pathogen effectors from the oomycete pathogen Phytophthora infestans, bacterial pathogen Ralstonia pseudosolanacearum, and the mirid bug Apolygus lucorum, suggesting that they may be critical virulence targets or components of the immune response. However, the biochemical activities of these proteins and how they contribute to plant health remain poorly defined. Here, we introduce the PUB-VI/RLCK-IXb clade in Arabidopsis, focusing on PUB32, PUB33, and PUB50. We show that PUB33 exhibits dual kinase and E3 ubiquitin ligase activities that are inversely regulated by autophosphorylation at Thr333. PUB33 forms homomers and heteromers with PUB32 which attenuate PUB33 catalytic activity. Although we did not observe clear defects in innate immune signaling in pub32, pub33, or pub50 mutants, we found that overexpression of PUB33 can suppress cell death triggered by the R. pseudosolanacearum effector RipV1 in N. benthamiana. Moreover, PUB33 directly ubiquitinates RipV1 in vitro and reduces RipV1 accumulation in planta, suggesting that it functions as part of the immune response against R. pseudosolanacearum.

African rice (Oryza glaberrima) was independently domesticated in West Africa around 3000 years ago, and has long been intertwined in the history of the region. The gradual replacement of African rice by Asian rice (Oryza sativa), which was introduced when European settlers arrived, has since dominated rice cultivation in Africa. Domesticated rice species are affected by bacterial leaf blight (BLB), which is caused by the pathogen Xanthomonas oryzae pv. oryzae (Xoo). Here we provide evidence that the bacterial leaf blight pathogen in Africa (AfXoo) belongs to a distinct phylogroup from the one circulating in Asia (AsXoo), and has a different evolutionary history. Analysis of 88 AfXoo genomes identified five groups, one of which is a highly diverse population that might have probably given rise to three independent clonal populations based on multiple genetic tests. Tip-dating analysis revealed that the emergence and expansion of AfXoo coincided with the rise and fall of African rice nearly a thousand years ago, and O. sativa served as a bottleneck in the evolution of AfXoo over time. Although the type III effectors (T3E), proteins that are secreted by the pathogen to evade host resistance or seize control of host nutrients, are highly conserved in AfXoo, we observed some variation in effector families. Different evolutionary modifications in the transcription activator-like effectors (TALEs), especially in repeat variable di-residues (RVDs), likely enabled adaptation to both host species. Previous analyses carried out on samples collected in Burkina Faso have shown that there could be more than one TALE repertoire combination in the field, and genome sequencing data revealed potential TALE evolutionary mechanisms that could happen. Our research provides a comprehensive genetic history of bacterial blight in West Africa, and its past and present impact on rice cultivation in the region. Author summaryFor thousands of years, rice cultivation has been an integral part of African agriculture. However, the cultivation of the locally domesticated African rice cultivar (Oryza glaberrima) has been gradually shifted towards Asian rice varieties (Oryza sativa), which has affected the adaptation of the native pathogen population. One of these pathogens is the causal agent of bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo). Here we performed a population genomics approach to understand the evolutionary history and virulence spectrum of African Xoo (AfXoo), a unique phylogroup within the Xanthomonas oryzae species. Our results suggest that AfXoo were first adapted to African rice at least a thousand years ago. The introduction of O. sativa has shaped the recent population dynamics of AfXoo. TALEs are tightly conserved in AfXoo with multiple sequence variations unique to different populations, which could be explained by different evolutionary forces acting upon both domesticated rice. Our results highlight the interplay between crop domestication and cultivation and pathogen evolution.

Cucurbit yellow vine disease (CYVD), caused by the bacterium Serratia ureilytica, is a phloem-associated disease of cucurbits. This study characterized the spatial and temporal distribution of S. ureilytica in Cucurbita pepo cultivar Delicata plants under greenhouse conditions using a GFP-tagged isolate (P01). Seedlings were sampled weekly for four weeks. Transverse sections from the stem, petiole, leaf, shoot apex, and root were imaged by laser scanning confocal and fluorescent dissecting microscopy. In parallel, bacterial abundance in each plant tissue was assessed by quantifying colony-forming units (CFU) via droplet plating over a 4-week time course. Across plant tissues and time points, S. ureilytica fluorescent signal was primarily concentrated in the inner and outer periphery of the bicollateral vascular bundles, with higher magnification images revealing mainly symplastic localization within phloem-associated cells. Consistent with the imaging results, bacterial quantification data showed a high abundance of CFUs in the main stem across weeks, with an irregular pattern of presence in the distal tissues at later time points. These results suggest that S. ureilytica is predominantly localized within phloem-associated cells and spreads both acropetally and basipetally during infection.

Isoflavone synthase (IFS), a cytochrome P450 monooxygenase of the CYP93C subfamily, catalyzes the conversion of flavanones into isoflavones, the first committed step in the biosynthesis of isoflavonoid phytoalexins. In pea (Pisum sativum L.), the phytoalexin pisatin plays a pivotal role in defense against pathogens. However, the molecular basis underlying IFS function in pea remains poorly understood. In this study, we performed a comprehensive genome-wide identification and characterization of IFS genes in pea. Three IFS candidates, PsIFS7A, PsIFS7B, and PsIFS7C, were identified that reside on chromosome 7, each harboring all conserved cytochrome P450 signature motifs. PsIFS genes exhibited predominant expression in root tissue, with transcript levels induced rapidly upon Aphanomyces euteiches infection. Enzymatic assays confirmed their catalytic activity in converting the flavanones naringenin and liquiritigenin into the isoflavones genistein and daidzein, respectively, both in vitro and in planta systems. Furthermore, all three PsIFS genes were found in close proximity to quantitative trait loci (QTL) associated with Aphanomyces root rot resistance. Together, these findings provide novel insights into the IFS gene family in pea and lay a foundation for metabolic engineering or molecular breeding strategies to enhance disease resistance through targeted modulation of pisatin biosynthesis.

Small RNAs (sRNAs) are key regulators of plant defense and have been implicated in cross-kingdom interactions with pathogens. The hemibiotrophic fungus Colletotrichum higginsianum infects Arabidopsis thaliana through three stages: appressorial penetration, biotrophy, and necrotrophy. However, the dynamics of fungal and plant sRNA populations across these three stages have not been elucidated. Using high-throughput sequencing, we profiled sRNAs from A. thaliana and C. higginsianum during in planta appressorium (PA), biotrophic (BP), and necrotrophic (NP) phases, and compared them to fungal mycelia (MY) and in vitro appressoria (VA). Our analyses revealed stage-specific patterns in sRNA accumulation in both the plant and the pathogen. In C. higginsianum, sRNAs were dominated by 29 nt species in PA, BP, MY, and VA, but shifted to 18 nt in NP, consistent with RNA degradation during host cell death. In A. thaliana, sRNAs transitioned from 30-33-nt in PA/BP to a 21 nt dominant peak in NP. Also, TE-derived siRNAs and other regulatory sRNAs (miRNAs, ncRNA, snoRNAs and snRNAs) declined during NP. A total of 62 host miRNAs showed differential accumulation, including core plant developmental regulators active across infection stages, and stage-specific miRNAs such as miR396, miR170/171, miR472, and miR858b. tRFs displayed opposite trends in host and pathogen: fungal tRFs declined in NP, while host misc-tRFs, 5'-tRFs, and 3'-tRFs increased, suggesting contrasting regulatory roles. These results provide new insights into RNA-mediated plant-fungal interactions.

Agrobacterium tumefaciens is an important plant pathogen, the causative agent of crown gall disease, and a foundational technology used for genetic transformation of plant tissue. More recently, A. tumefaciens has been adopted as a genetically tractable model organism for studying bacterial cell cycle regulation, developmental pathways, and niche construction. The transition from a free-living bacterium within the rhizosphere to association with a plant host and subsequent transformation of plant tissue is one aspect of A. tumefaciens life history that encompasses all three of these processes. Such events are coordinated by multiple regulatory modules which must sense external and/or internal cues, integrate these inputs, and effect appropriate changes in gene expression and cellular responses to maximize fitness. Here, we evaluate the contribution of the two-component system, FeuP-FeuQ, to gene expression and developmental phenotypes including surface attachment/biofilm formation, swimming motility, and tumorigenesis. feuPQ operon organization suggests translational coupling during expression of the response regulator, FeuP (Atu0970), and sensor kinase, FeuQ (Atu0971). In-frame, non-polar deletion of feuP or feuQ individually, or the entire feuPQ operon, resulted in reduced biofilm formation, swimming motility, and tumor formation, without adversely affecting planktonic growth. Transcriptomic profiling identified [~]300 differentially expressed genes when the feuPQ locus was disrupted, including genes affecting flagellar motility, succinoglycan production, and type VI secretion. Phenotype profiling emphasized the contribution of feuPQ to withstanding osmotic, ionic, and antimicrobial stressors. Together, these data highlight FeuPQ as a global regulator of cellular responses which likely contribute to overall fitness during rhizosphere lifestyle transitions. IMPORTANCEAgrobacterium tumefaciens is an important plant pathogen able to genetically transform numerous plant species. In the rhizosphere, this bacterium encounters many challenges ranging from antagonistic and competing microbes, to plant host defenses, to rapidly changing environmental conditions. Efficient host interaction leading to plant transformation requires coordination of bacterial motility, attachment, and defense mechanisms, among other processes. This work identifies two proteins, FeuP and FeuQ, that together contribute to such coordination. The importance of this work is in identifying the FeuPQ system as a global regulator of these and other processes, possibly enabling targeted interventions to promote or inhibit plant transformation.

Sustaining high agricultural productivity with minimal environmental impact requires innovative and sustainable strategies that reduce reliance on mineral fertilizers. Promoting root association with plant growth-promoting bacteria (PGPB), either within the native microbiome or through bioinoculant application, represents a promising strategy to improve crop performance while reducing mineral fertilizer inputs. The success of this strategy, however, is strongly influenced by plant genetic traits that regulate microbial recruitment and colonization. Here, we tested whether silencing the ABAP1 Interacting Protein (AIP10), a negative regulator that links cell division with primary metabolism, modulates the association of Arabidopsis thaliana to PGPB. Non-inoculated aip10-1 roots exhibited gene expression patterns similar to genotypes with enhanced microbial associations. AIP10 silencing reshaped root and rhizosphere bacterial communities, favoring beneficial PGPB associations and limiting potential pathogens. Consistently, aip10-1 plants showed greater colonization by inoculated diazotrophic PGPB, particularly in low fertilization conditions, leading to increased plant performance. These effects were accompanied by modulation of plant cell cycle and nitrogen assimilation pathways, together with increased bacterial colonization and nifH expression. Our findings suggest that AIP10 functions as a regulatory hub coordinating growth and metabolism with beneficial PGPB recruitment. Modulating AIP10 could enhance plant productivity and support more sustainable and regenerative agriculture practices. HighlightAIP10 silencing participates in a regulatory hub coordinating plant cell cycle and metabolism with recruitment of beneficial bacteria in the root microbiota, contributing to improved plant growth and productivity under nutrient-limited conditions.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are essential regulators of plant growth, development, and stress adaptation. In this study, we performed a comprehensive genome-wide identification of SNARE genes in cucumber (Cucumis sativus L.), uncovering 51 putative members designated as CsSNAREs. Phylogenetic analysis confirmed that these genes cluster into five major clades: Qa-CsSNARE (14), Qb-CsSNARE (9), Qc-CsSNARE (10), Qb+c-CsSNARE (3), and R-CsSNARE (15). Bioinformatic analysis of their promoter regions, coupled with expression profiling under diverse abiotic stress conditions, highlighted a heightened responsiveness within the Qa-CsSNARE subfamily. To validate this, we selected representative Qa-CsSNARE genes for quantitative real-time PCR analysis under drought and salt stress. Among these, CsSYP121 was notably induced by salt treatment. We subsequently generated transgenic cucumber lines overexpressing CsSYP121 and challenged them with salinity. Phenotypic assessment, combined with measurements of reactive oxygen species (ROS) accumulation and K+/Na+ ratios, demonstrated that CsSYP121 overexpression (OE) confers enhanced salt tolerance and boosts antioxidant capacity. We propose a model wherein CsSYP121 mitigates ROS-induced cellular damage under salt stress, potentially through promoting K+/Na+ homeostasis, thereby improving plant performance under saline conditions. Our findings identify CsSYP121 as a promising candidate gene for breeding salt-tolerant crops.

Belowground, plants are exposed to a wide range of biotic stresses that vary in severity and nature, including tissue damage, disruption of vascular connectivity, and depletion of assimilates. How plants adapt their root systems to cope with different types of belowground biotic stresses is not well known. In this paper we compare above- and belowground plant adaptations to three nematode species with distinct tissue migration and feeding behaviours to study mechanisms underlying tolerance to different types of biotic stresses. We monitored both green canopy growth and changes in root system architecture of Arabidopsis inoculated with Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita. This revealed three distinct phases in aboveground plant responses: (i) initial growth inhibition associated with host invasion and tissue damage, (ii) persistent growth reduction associated with nematode sedentarism, and (iii) late growth stimulus in more advanced stages of infection. Specific adaptations in the root systems further revealed fundamentally different stress coping strategies. Tissue damage and intermittent feeding by P. penetrans in the root cortex did not induce significant changes in root system architecture. Tissue damage to the root cortex and prolonged feeding on host vascular cells by H. schachtii induced secondary root formation compensating for primary root growth inhibition. Prolonged feeding on host vascular cell by M. incognita alone did not induce secondary root formation, but was accompanied by typical local tissue swelling instead. Our data suggest that local secondary root formation and tissue swelling are two distinct compensatory mechanisms underlying tolerance to sedentarism by root-feeding nematodes. HighlightHow plants utilize root system plasticity to cope with different types of biotic stresses by root feeding nematodes remains largely unknown. Here, we report on specific adaptive growth responses in Arabidopsis roots to three nematode species, Pratylenchus penetrans, Heterodera schachtii, and Meloidogyne incognita, with fundamentally different strategies for host invasion, subsequent migration through host tissue, and feeding on host cells.