Sara Franco Ortega

A mung bean infection model has previously been shown to differentiate between non-virulent and virulent Pseudomonas aeruginosa bacteria. However, it remains unclear how plant and bacteria adjust their gene expression during infection and whether the mung bean model can be used to compare the virulence of clinical cystic fibrosis (CF) P. aeruginosa lung isolates. Here, we first explored temporal transcriptomics of P. aeruginosa PAO1 and mung bean during an infection. We found that bacterial gene expression followed temporal changes, with an increase in the expression of O-antigen biosynthetic genes, chemotaxis, phosphate intake and phenazine production. Mung bean responded by upregulating genes associated with defence mechanisms and downregulating genes involved in the plant development. From the PAO1 perspective, the core-transcriptomic responses in the mung bean were similar to its responses previously observed in wound and excision and in in vitro media and sputum models, while differed from those observed in the bronchial cell model. Furthermore, we used the mung bean to assess the virulence of 119 clinical P. aeruginosa CF strains originating from the Copenhagen CF clinic. By quantifying bacterial virulence as a reduction in root and shoot growth and weight of the seeds, we found that CF strains isolated at later compared to early stages of lung infections showed higher virulence. This difference corresponded with the higher number of immune modulation-associated virulence genes and lower number of motility and effector genes, present in the genomes of late compared to early isolated CF strains.

In-host bacterial evolution presents a major barrier to effective infection management, driving phenotypic adaptations such as antibiotic resistance and altered virulence. Pseudomonas aeruginosa, a key opportunistic pathogen, frequently undergoes rapid evolutionary changes during chronic lung infections, complicating diagnosis and treatment. Current strain typing via whole genome sequencing or selective culturing is costly and time-intensive, and the complex relationship between genetic variations and the resulting phenotype makes clinically relevant pathotypes difficult to identify. Here, we report a cross-reactive, glycopolymer-based fluorescent sensor array capable of directly identifying phenotypic changes related to in-host evolution in P. aeruginosa. The sensor array can accurately distinguish phenotypic variations arising from single-gene defects and discriminate clinical isolates with known differences in their evolutionary and pathoadaptive trajectories. Notably, our system is also capable of identifying P. aeruginosa isolates as distinct from other bacterial species commonly found in complex polymicrobial lung infections. Our modular platform presents an opportunity to develop sensor arrays that target carbohydrate recognition in a variety of pathogens, offering potential application as a rapid diagnostic tool to inform clinical treatment decisions based on the direct classification of phenotypic profiles.

To understand why close wild plant relatives of crops, such as Solanum dulcamara, are resistant to Ralstonia solanacearum we need genome resources to perform comparative studies and identify key genes and pathways. We de-novo assembled and annotated the genome of a resistant/tolerant S. dulcamara and susceptible Solanum nigrum plant using a hybrid approach including Oxford Nanopore Technologies and Illumina sequencing. Comparative genomic analysis was then performed to find differences between the genome of S. dulcamara and other susceptible Solanaceous species including potato, tomato, aubergine, and S. nigrum and one susceptible and one resistant S. americanum accession. We identified genes associated with auxin-transport only in S. dulcamara and a collection of pattern recognition receptors (PRRs) in orthogroups only found in resistant/tolerant plant species, which we hypothesize may improve recognition of pathogen-associated molecular patterns associated with R. solanacearum. We also identified an arsenal of nucleotide-binding leucine-rich repeat receptors (NLRs) in the S. nigrum genome that are shared with the other susceptible species and could be acting as susceptibility factors. Finally, we identified differences in methylation frequency across the gene bodies in both species, which may be associated with epigenetic regulation of resistance. Future work should assess the functional role of these PRRs and NLRs during bacterial wilt development to determine if they could offer potential novel targets for breeding to improve bacterial wilt resistance.

Ash dieback caused by the fungus Hymenoscyphus fraxineus has devastated the European ash tree population since it arrived in Europe in 1992. Great effort has been put into breeding programs to increase the genetic diversity of ash trees and find heritable genetic markers associated with resistance, or tolerance mechanisms, to ash dieback. To facilitate identification of molecular markers, we used Oxford Nanopore Technologies combined with Illumina sequencing to obtain an accurate and contiguous ash genome. We used this genome to reanalyze transcriptome data from a Danish ash panel of 182 tree accessions. Using associative transcriptomics, we identified 175 gene expression markers, including 11 genes annotated as dormancy MADS-box transcription factors which are associated with ash bud dormancy, flowering, and senescence. We hypothesize that tolerant trees both break dormancy earlier in the year by increasing the expression of flowering-related SOC1 MADS-box and reducing the expression of SVP-like MADS-box, whilst also accelerating senescence by increasing the expression of JOINTLESS MADS-box genes. DNA methylation differences in the promoters of MADS-box genes between 1 tolerant and 1 susceptible tree indicate potential epigenetic regulation of these traits.

To understand why close wild plant relatives of crops, such as Solanum dulcamara , are resistant to Ralstonia solanacearum we need genetic resources to perform comparative studies and identify key genes and pathways. We de-novo assembled and annotated the genome of resistant S. dulcamara and susceptible Solanum nigrum plants using a hybrid approach including Oxford Nanopore Technologies and Illumina sequencing. Comparative genomic analysis was then performed to find differences between the genome of S. dulcamara and other susceptible Solanaceous species including potato, tomato, aubergine, and S. nigrum and one susceptible and one resistant S. americanum accession. We identified genes associated with auxin-transport only in S. dulcamara and a collection of pattern recognition receptors (PRRs) was identified in orthogroups only found in plant species with resistant/tolerant phenotype, suggesting novel plant receptors in these accessions that may improve recognition of pathogen-associated molecular patterns (PAMPs) associated with R. solanacearum . We also identified differences in methylation frequency across the gene bodies in both species, which may be associated with epigenetic regulation of resistance. Future work should assess the functional role of these PRRs during bacterial wilt development to determine if they could offer potential novel targets for breeding improved wilt resistance.

Ash dieback caused by the fungus Hymenoscyphus fraxineus has devastated the European ash tree population since it arrived in Europe in 1992. Great effort has been put into breeding programmes to increase the genetic diversity of ash trees and find heritable genetic markers associated with resistance, or tolerance mechanisms, to ash dieback. To facilitate identification of molecular markers, we used Oxford Nanopore Technologies combined with Illumina sequencing to obtain an accurate and contiguous ash genome. We used this genome to reanalyse transcriptome data from a Danish ash panel of 182 tree accessions. Using associative transcriptomics, we identified 175 gene expression markers (GEMs), including 11 genes annotated as dormancy MADS-box transcription factors which are associated with ash bud dormancy, flowering and senescence. We hypothesize that tolerant trees both break dormancy earlier in the year by increasing the expression of flowering-related SOC1 MADS-box and reducing the expression of SVP-like MADS-box, whilst also accelerating senescence by increasing the expression of JOINTLESS MADS-box genes. DNA methylation differences in the promoters of MADS-box genes between one tolerant and one susceptible tree indicate potential epigenetic regulation of these traits. Article Summary: Ash dieback has devastated European ash tree populations. To aid in breeding programmes focused on finding solutions against this pathogen, we have assembled a new ash genome. This new genome helped us to identify genes related to tree biological life cycles, expressed differently in tolerant and susceptible trees. For the first time, we have also discovered that susceptible and tolerant trees showed different DNA methylation frequencies in those genes, suggesting epigenetic regulation. DNA methylation can turn on/off gene expression without changing the DNA sequence. These genes, and their regulatory elements, are ideal targets during breeding programmes combating this pathogen.

Phages can successfully be used in vitro and in planta to biocontrol the phytopathogenic Ralstonia solanacearum bacterium —the causal agent of bacterial wilt disease. However, phage biocontrol outcomes are still variable, and it is unclear what causes this. In this study, we assessed the efficiency of four phages in controlled in vitro and in planta experiments in all one‐ and two‐phage combinations. We found that using phages in combination did not improve the phage biocontrol efficiency relative to single phage treatments, while certain phages and their combinations were more effective than the others. High intra‐treatment variability in phage efficiency was observed across all phage treatments, which was associated with clear shifts in microbiome composition, a reduction in R. solanacearum and an increase in phage densities. We further identified the bacterial taxa that were associated with these ‘shifted’ microbiomes and conducted additional plant growth experiments, demonstrating that some of the enriched bacterial species could protect plants from R. solanacearum infections—a pattern which was also observed using partial least squares path modelling (PLS‐PM). Together, these results suggest that phages could open niche space for beneficial bacteria by reducing pathogen densities and that variability in phage biocontrol outcomes is rhizosphere microbiome‐dependent, which can introduce between‐replicate variation, even in controlled greenhouse conditions.

This study begins by examining phenotypic variations in field growth among four parental Vietnamese rice lines, consisting of two Indica (PD211/GL37) and two Japonica (J23/SRA2-1) cultivars, which differ in life-cycle durations. Their phenotypic observations revealed both similarities and differences in growth patterns and field responses, setting the stage for further genomic investigation. We then focused on the sequencing and de novo genome assembly of these lines using high-coverage Illumina sequencing and achieving pseudochromosome assemblies ranging between 379 Mbp and 384 Mbp. The assemblies were further enhanced by annotation processes, designating between 44,427 and 48,704 gene models/genome. A comparative genomic analysis revealed that the Japonica varieties (J23/SRA2-1) exhibited more genetic similarity than the Indica varieties (PD211/GL37). From this, a phylogenetic analysis on the phytochrome C (phyC) gene distinctly positions the Indica and Japonica lines within their respective clades, affirming their genetic diversity and lineage accuracy. These genomic resources will pave the way for identifying quantitative trait loci (QTLs) critical for developing rice cultivars with shorter life cycles, thus enhancing resilience to adverse climatic impacts in Vietnam. This study provides a foundational step towards leveraging genomic data for rice breeding programs aimed at ensuring food security in the face of climate change.

Summary: Bacterial pathogens inject effector proteins inside plant cells to manipulate cellular functions and achieve a successful infection. The soil‐borne pathogen Ralstonia solanacearum (Smith), the causal agent of bacterial wilt disease, secretes > 70 different effectors inside plant cells, although only a handful of them have been thoroughly characterized. One of these effectors, named RipI, is required for full R. solanacearum pathogenicity. RipI associates with plant glutamate decarboxylases (GADs) to promote the accumulation of gamma‐aminobutyric acid (GABA), which serves as bacterial nutrient. In this work, we found that RipI can also suppress plant immune responses to bacterial elicitors, which seems to be unrelated to the ability of RipI to induce GABA accumulation and plant cell death. A detailed characterization of the RipI features that contribute to its virulence activities identified two residues at the C‐terminal domain that mediate RipI interaction with plant GADs and the subsequent promotion of GABA accumulation. These residues are also required for the appropriate homeostasis of RipI in plant cells and the induction of cell death, although they are partially dispensable for the suppression of plant immune responses. Altogether, we decipher and uncouple the virulence activities of an important bacterial effector at the biochemical level.

The airborne mycobiota has been understudied in comparison with the mycobiota present in other agricultural environments. Traditional, culture-based methods allow the study of a small fraction of the organisms present in the atmosphere, thus missing important information. In this study, the aerial mycobiota in a rice paddy has been examined during the cropping season (from June to September 2016) using qPCRs for two important rice pathogens (Pyricularia oryzae and Bipolaris oryzae) and by using DNA metabarcoding of the fungal ITS region. The metabarcoding results demonstrated a higher alpha diversity (Shannon–Wiener diversity index H′ and total number of observed species) at the beginning of the trial (June), suggesting a higher level of community complexity, compared with the end of the season. The main taxa identified by HTS analysis showed a shift in their relative abundance that drove the cluster separation as a function of time and temperature. The most abundant OTUs corresponded to genera such as Cladosporium, Alternaria, Myrothecium, or Pyricularia. Changes in the mycobiota composition were clearly dependent on the average air temperature with a potential impact on disease development in rice. In parallel, oligotyping analysis was performed to obtain a sub-OTU identification which revealed the presence of several oligotypes of Pyricularia and Bipolaris with relative abundance changing during monitoring.