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Oomycetes are filamentous organisms that resemble fungi in terms of morphology and life cycle, primarily due to convergent evolution. The success of pathogenic oomycetes lies in their ability to adapt and overcome host resistance, occasionally transitioning to new hosts. During plant infection, these organisms secrete effector proteins and other compounds during plant infection, as a molecular arsenal that contributes to their pathogenic success. Genomic sequencing, transcriptomic analysis, and proteomic studies have revealed highly diverse effector repertoires among different oomycete pathogens, highlighting their adaptability and evolution potential. The obligate biotrophic oomycete Plasmopara viticola affects grapevine plants ( Vitis vinifera L.) causing the downy mildew disease, with significant economic impact. This disease is devastating in Europe, leading to substantial production losses. Even though Plasmopara viticola is a well-known pathogen, to date there are scarce reviews summarising pathogenicity, virulence, the genetics and molecular mechanisms of interaction with grapevine. This review aims to explore the current knowledge of the infection strategy, lifecycle, effector molecules, and pathogenicity of Plasmopara viticola . The recent sequencing of the Plasmopara viticola genome has provided new insights into understanding the infection strategies employed by this pathogen. Additionally, we will highlight the contributions of omics technologies in unravelling the ongoing evolution of this oomycete, including the first in-plant proteome analysis of the pathogen.

In yeast and animal cells, phosphatidylinositol‐3‐monophosphate 5‐kinases produce phosphatidylinositol (3,5)‐bisphosphate (PtdIns(3,5)P₂) and have been implicated in endomembrane trafficking and pH control in the vacuole. In plants, PtdIns(3,5)P₂ is synthesized by the Fab1 family, four orthologs of which exist in Arabidopsis: FAB1A and FAB1B, both from the PIKfyve/Fab1 family; FAB1C and FAB1D, both without a PIKfyve domain and of unclear role. Using a reverse genetics and cell biology approach, we investigated the function of the Arabidopsis genes encoding FAB1B and FAB1D, both highly expressed in pollen. Pollen viability, germination and tube morphology were not significantly affected in homozygous mutant plants. In vivo, mutant pollen fertilized ovules leading to normal seeds and siliques. The same result was obtained when mutant ovules were fertilized with wild‐type pollen. Double mutant pollen for the two genes was able to fertilize and develop plants no different from the wild‐type. At the cellular level, fab1b and fab1d pollen tubes were found to exhibit perturbations in membrane recycling, vacuolar acidification and decreased production of reactive oxygen species (ROS). Subcellular imaging of FAB1B‐GFP revealed that the protein localized to the endomembrane compartment, whereas FAB1D‐GFP localized mostly to the cytosol and sperm cells. These results were discussed considering possible complementary roles of FAB1B and FAB1D.

Allene oxide synthase (AOS) is a key enzyme of the jasmonic acid (JA) signaling pathway. The AOS gene was previously found to be upregulated in an Asian chestnut species resistant to infection by the oomycete Phytophthora cinnamomi ( Castanea crenata ), while lower expression values were detected in the susceptible European chestnut ( Castanea sativa ). Here, we report a genetic and functional characterization of the C. crenata AOS (CcAOS) upon its heterologous gene expression in a susceptible ecotype of Arabidopsis thaliana , which contains a single AOS gene. It was found that Arabidopsis plants expressing CcAOS delay pathogen progression and exhibit more vigorous growth in its presence. They also show upregulation of jasmonic acid and salicylic acid-related genes. As in its native species, heterologous CcAOS localized to plastids, as revealed by confocal imaging of the CcAOS-eGFP fusion protein in transgenic Arabidopsis roots. This observation was confirmed upon transient expression in Nicotiana benthamiana leaf epidermal cells. To further confirm a specific role of CcAOS in the defense mechanism against the pathogen, we performed crosses between transgenic CcAOS plants and an infertile Arabidopsis AOS knockout mutant line. It was found that plants expressing CcAOS exhibit normal growth, remain infertile but are significantly more tolerant to the pathogen than wild type plants. Together, our results indicate that CcAOS is an important player in plant defense responses against oomycete infection and that its expression in susceptible varieties may be a valuable tool to mitigate biotic stress responses.

Castanea sativa , a species of high ecological and economic relevance in Europe, faces severe threats from root rot caused by Phytophthora cinnamomi . To explore genetic strategies for enhancing disease tolerance, we investigated the functional role of a chestnut gene homologous to Ginkgo biloba ’s ginkbilobin-2 ( Cast_Gnk2-like ), known for its antifungal properties. Using Agrobacterium tumefaciens -mediated transformation, the Cast_Gnk2-like gene was introduced into somatic embryos from two embryogenic chestnut lines. Transformation efficiency was genotype-dependent, and varied from 14.2% to 2.5%. Twelve independent transgenic lines were confirmed by PCR, and each was estimated to carry a single copy of the transgene. Gene expression analysis revealed significant Cast_Gnk2-like transcript levels in two transgenic lines. Following cold storage and germination treatment, viable transgenic plants were regenerated. Disease tolerance assays demonstrated that Cast_Gnk2-like overexpression significantly reduced root necrosis and symptom severity, indicating enhanced tolerance to P. cinnamomi . These findings highlight the potential of targeted gene overexpression to improve disease resilience in chestnut through genetic engineering.

In recent decades an extensive mortality and decline of Quercus suber populations mainly caused by Phytophthora cinnamomi has been observed. In the current study, a chestnut gene homologous to ginkbilobin-2 (Cast_ Gnk2-like ), which in Ginkgo biloba codifies an antifungal protein, was transferred into cork oak somatic embryos of three different embryogenic lines by Agrobacterium mediated transformation. The transformation efficiency varied on the genotype from 2.5 to 9.2%, and a total of 22 independent transformed lines were obtained. The presence of Cast_Gnk2-like gene in transgenic embryos was verified in all lines by PCR. The number of transgene copies was estimated by qPCR in embryogenic lines with high proliferation ability and it varied between 1 and 5. In addition, the expression levels of Cast_Gnk2-like gene were determined in the embryogenic lines, with higher levels in lines derived from the genotype ALM6-WT. Transgenic plants were obtained from all transgenic lines and evaluated after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In vitro tolerance tests made under controlled conditions and following zoospore treatment showed that plants overexpressing Cast_Gnk2-like gene improved tolerance against Pc when compared to wild type ones.

Brain metastases (BM) critically reduce breast cancer (BC) patients’ survival. Extravasation is pivotal for BM development, but the underlying events at the blood-brain barrier (BBB) remain elusive. We aimed to unravel the players and mechanisms governing BC cells (BCCs)-BBB interaction. For that, mixed cultures of human brain microvascular endothelial cells (HBMECs), mimicking the BBB, and brain-tropic triple-negative BCCs (MDA-MB-231 Br4), or non-brain-tropic (MDA-MB-231) or non-metastatic cells (MCF-7) were established. Temporal and spatial analysis of BCCs-BBB interactions (live-cell imaging automated microscopy), and assessments of endothelial-to-mesenchymal transition (EndMT) markers, transcription factors, cytoskeletal proteins, and morphology (immunocytochemistry) were performed. BBB integrity (permeability, transendothelial electrical resistance) and endothelial migration (wound-healing) were also assessed. Our results revealed that contrasting with non-metastatic and non-brain-tropic cells, BCCs quickly developed an invasive, migratory phenotype, characterized by invadopodium formation and reduced roundness. Spatial analysis showed different positioning of BCCs relative to the BBB endothelium over time, with 14% of BCCs transmigrated after 3 h, compromising BBB integrity through endothelial holes, reduced tightness, and increased permeability. Prior to transmigration, alterations in adhesion markers (E-selectin, ICAM-1, CD24, CD34, β3-integrin, Sialyl-Lewis X) were observed. EndMT was also evident by decreased endothelial (β-catenin and pan cytokeratin) and increased mesenchymal (vimentin, neuronal-cadherin, Slug, ZEB1) markers, elongation (RhoA, α-SMA), nuclear deformation, and migratory capacity. Caveolin-1 silencing in HBMEC decreased BCCs transmigration. This study reveals significant BBB phenotypic and structural changes, facilitating both paracellular and transcellular BCCs transmigration. These findings provide advanced understanding of BCCs trafficking across the BBB, aiding strategy development to prevent extravasation and BM.

Plasmopara viticola , an obligate biotrophic oomycete, is the causal agent of one of the most harmful grapevine diseases, downy mildew. Within this pathosystem, much information is gathered on the host, as characterization of pathogenicity and infection strategy of a biotrophic pathogen is quite challenging. Molecular insights into P. viticola development and pathogenicity are just beginning to be uncovered, mainly by transcriptomic studies. Plasmopara viticola proteome and secretome were only predicted based on transcriptome data. In this study, we have identified the in-planta proteome of P. viticola during infection of a susceptible (‘Trincadeira’) and a Rpv3 -mediated resistance (‘Regent’) grapevine cultivar. Four hundred and twenty P. viticola proteins were identified on a label-free mass spectrometry-based approach of the apoplastic fluid of grapevine leaves. Overall, our study suggests that, in the compatible interaction, P. viticola manipulates salicylic-acid pathway and isoprenoid biosynthesis to enhance plant colonization. Furthermore, during the incompatible interaction, development-associated proteins increased while oxidoreductases protect P. viticola from ROS-associated plant defence mechanism. Up to our knowledge this is the first in-planta proteome characterization of this biotrophic pathogen, thus this study will open new insights into our understanding of this pathogen colonization strategy of both susceptible and Rpv3 -mediated resistance grapevine genotypes.

Breast cancer brain metastases (BCBMs) have been underinvestigated despite their high incidence and poor outcome. MicroRNAs (miRNAs), and particularly circulating miRNAs, regulate multiple cellular functions, and their deregulation has been reported in different types of cancer and metastasis. However, their signature in plasma along brain metastasis development and their relevant targets remain undetermined. Here, we used a mouse model of BCBM and next‐generation sequencing (NGS) to establish the alterations in circulating miRNAs during brain metastasis formation and development. We further performed bioinformatics analysis to identify their targets with relevance in the metastatic process. We additionally analyzed human resected brain metastasis samples of breast cancer patients for target expression validation. Breast cancer cells were injected in the carotid artery of mice to preferentially induce metastasis in the brain, and samples were collected at different timepoints (5 h, 3, 7, and 10 days) to follow metastasis development in the brain and in peripheral organs. Metastases were detected from 7 days onwards, mainly in the brain. NGS revealed a deregulation of circulating miRNA profile during BCBM progression, rising from 18% at 3 days to 30% at 10 days following malignant cells’ injection. Work was focused on those altered prior to metastasis detection, among which were miR‐802‐5p and miR‐194‐5p, whose downregulation was validated by qPCR. Using targetscan and diana tools, the transcription factor myocyte enhancer factor 2C (MEF2C) was identified as a target for both miRNAs, and its expression was increasingly observed in malignant cells along brain metastasis development. Its upregulation was also observed in peritumoral astrocytes pointing to a role of MEF2C in the crosstalk between tumor cells and astrocytes. MEF2C expression was also observed in human BCBM, validating the observation in mouse. Collectively, downregulation of circulating miR‐802‐5p and miR‐194‐5p appears as a precocious event in BCBM and MEF2C emerges as a new player in brain metastasis development. The search for aberrantly expressed miRNAs in plasma along breast cancer brain metastasis (BCBM) development revealed miR‐802‐5p and miR‐194‐5p downregulation as an early event. MEF2C emerged as their relevant target, with an increasing expression along BCBM expansion and in resected brain metastasis from breast cancer patients. Such miRNAs and MEF2C appear as early events and new players in BCBM development.

The Arabidopsis thaliana genome contains 20 CNGCs, which are proposed to encode cyclic nucleotide gated, non-selective, Ca²⁺-permeable ion channels. CNGC7 and CNGC8 are the two most similar with 74% protein sequence identity, and both genes are preferentially expressed in pollen. Two independent loss-of-function T-DNA insertions were identified for both genes and used to generate plant lines in which only one of the two alleles was segregating (e.g., cngc7-1+/-/cngc8-2-/- and cngc7-3-/-/cngc8-1+/-). While normal pollen transmission was observed for single gene mutations, pollen harboring mutations in both cngc7 and 8 were found to be male sterile (transmission efficiency reduced by more than 3000-fold). Pollen grains harboring T-DNA disruptions of both cngc7 and 8 displayed a high frequency of bursting when germinated in vitro. The male sterile defect could be rescued through pollen expression of a CNGC7 or 8 transgene including a CNGC7 with an N-terminal GFP-tag. However, rescue efficiencies were reduced ∼10-fold when the CNGC7 or 8 included an F to W substitution (F589W and F624W, respectively) at the junction between the putative cyclic nucleotide binding-site and the calmodulin binding-site, identifying this junction as important for proper functioning of a plant CNGC. Using confocal microscopy, GFP-CNGC7 was found to preferentially localize to the plasma membrane at the flanks of the growing tip. Together these results indicate that CNGC7 and 8 are at least partially redundant and provide an essential function at the initiation of pollen tube tip growth.