Sebastian Pfeilmeier

Plant‐pathogenic bacteria colonise their hosts using various strategies, exploiting both natural openings and wounds in leaves and roots. The vascular pathogen Xanthomonas campestris pv. campestris (Xcc) enters its host through hydathodes, organs at the leaf margin involved in guttation. Subsequently, Xcc breaches the hydathode–xylem barrier and progresses into the xylem vessels causing systemic disease. To elucidate the mechanisms that underpin the different stages of an Xcc infection, a need exists to image bacterial progression in planta in a non‐invasive manner. Here, we describe a phenotyping setup and Python image analysis pipeline for capturing 16 independent Xcc infections in Arabidopsis thaliana plants in parallel over time. The setup combines an RGB camera for imaging disease symptoms and an ultrasensitive CCD camera for monitoring bacterial progression inside leaves using bioluminescence. The method reliably quantified bacterial growth in planta for two bacterial species, that is, vascular Xcc and the mesophyll pathogen Pseudomonas syringae pv. tomato (Pst). The camera resolution allowed Xcc imaging already in the hydathodes, yielding reproducible data for the first stages prior to the systemic infection. Data obtained through the image analysis pipeline was robust and validated findings from other bioluminescence imaging methods, while requiring fewer samples. Moreover, bioluminescence was reliably detected within 5 min, offering a significant time advantage over our previously reported method with light‐sensitive films. Thus, this method is suitable to quantify the resistance level of a large number of Arabidopsis thaliana accessions and mutant lines to different bacterial strains in a non‐invasive manner for phenotypic screenings.

To colonize plants, pathogenic bacteria modulate the biology of the host employing different bacterial secretion systems. For example, the type II secretion system (T2SS) releases toxins, proteases, lipases and carbohydrate-degrading enzymes into the extracellular environment to promote tissue softening and soft rot. In this way, the T2SS promotes virulence of phytopathogenic Gram-negative bacteria. However, the role of the T2SS and its substrates for vascular disease remains enigmatic. Here, we show that the Xps-T2SS allows Xanthomonas campestris pv. campestris (Xcc) to breach the tissue barrier between hydathodes– the initial bacterial entry point – and xylem thereby gaining access to the leaf vasculature. Yet, Xps-T2SS was dispensable for bacterial multiplication in the leaf apoplast or inside the hydathode cavity suggesting a role beyond plant defense suppression or nutrient acquisition. Using comparative genomics, four plant cell wall-degrading enzymes (CWDEs) were found to be associated with vascular pathogenesis. Testing gene knockout combinations of those enzymes revealed that virulence of only the quadruple CWDE mutant was down to the level of the xps -T2SS mutant. Our results thus demonstrate that the Xps-T2SS and a set of CWDEs that is likely secreted by this system allow Xcc to break this tissue barrier enabling long-distance mobility of Xcc inside the host plant. We thus expand our understanding on how certain bacterial pathogens have specialized towards vascular pathogens.

Plant pathogenic bacteria use various entry strategies to colonize their host, like entering through natural openings and wounds in leaves and roots. The vascular pathogen Xanthomonas campestris pv. campestris (Xcc) enters through hydathodes, organs at the leaf margin involved in guttation. Subsequently, Xcc breaks out from infected hydathodes, progressing into the xylem vessels and causing systemic disease. To elucidate the mechanisms that underpin the different stages of Xcc pathogenesis, a need exists to image Xcc progression in planta in a non-invasive manner. Here, we describe a phenotyping setup and Python image analysis pipeline capturing the Xcc infection in 16 Arabidopsis thaliana plants in parallel over time. The setup used both an RGB to capture disease symptoms and an ultra-sensitive CCD camera to monitor bacterial progression inside the leaves using bioluminescence. We demonstrate that the image analysis pipeline reliably quantifies bacterial growth in planta for two bacterial species, that is vascular Xcc and the mesophyll pathogen Pseudomonas syringae pv. tomato. The resolution of the camera allowed early detection of Xcc in the hydathodes, yielding valuable information on this early stage of the Xcc infection process. The data obtained through the automated image analysis pipeline was robust and validated findings from other bioluminescence imaging methods, while requiring fewer samples. We can thus quantify the resistance level of a large number of Arabidopsis thaliana accessions and mutant lines to different bacterial strains in a non-invasive manner for phenotypic screenings.

Bacterial plant pathogens exploit natural openings, such as pores or wounds, to enter the plant interior and cause disease. Plants guard these openings through defense mechanisms. However, bacteria from the genus Xanthomonas have specialized in that they enter their host via a special entry point, the hydathode—an organ at the leaf margin involved in xylem sap guttation. Hydathodes can mount an immune response against bacteria, including non-adapted and adapted pathogens like X. campestris pv. campestris (Xcc) that cause vascular disease. Previously, it was shown that the RKS1/ZAR1 immune complex confers vascular resistance against Xcc by recognizing XopAC activity, a type III effector (T3E). However, in absence of XopAC recognition, Arabidopsis Col-0 hydathodes still display resistance against Xcc. Here we mapped the causal gene using an inoculation method that promotes Xcc hydathode entry. Using a population of Recombinant Inbred Lines (RILs) of a cross between a susceptible (Oy-0) and resistant accession (Col-0), a major QTL for Xcc resistance was found on the right arm of Chromosome 5 in Col-0. Combining this result with a genome-wide association analysis yielded a single candidate gene encoding a coiled-coil nucleotide-binding leucine-rich repeat (CNL-type) immune receptor protein called SUPPRESSOR OF TOPP4 1 (SUT1). Expression of SUT1 was confirmed in hydathodes. We reveal that RKS1/ZAR1 and SUT1 confer different levels of Xcc resistance in different tissue types. Both RKS1/ZAR1 and SUT1 are alone sufficient for Xcc resistance in Col-0 hydathodes. However, RKS1/ZAR1 resistance is also effective in tissue types that represent late infection stages, i.e. xylem and mesophyll. In contrast, SUT1 resistance is not effective in the xylem, while weakly additive to RKS1/ZAR1 in the mesophyll. We thus identify a novel R gene, SUT1 , that confers Xcc resistance primarily early in the infection during hydathode colonization. Author summary: Black rot disease, caused by the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc), is an economically important disease of cabbage crops. Xcc is unique in that it enters the plant interior through specialized organs at the leaf margin. These structures called hydathodes contain water pores and are involved in root pressure regulation. Although we know that hydathodes can mount an immune response against these bacteria, specific immune receptors still need to be discovered. Here we use the model plant Arabidopsis thaliana to map an hydathode-effective resistance gene. By screening two different Arabidopsis populations, we could map a single gene, SUT1, that is involved in this resistance. SUT1 restricts the early hydathode colonization by Xcc thus suppressing disease progression. Interestingly, SUT1 resistance was not effective in the plant vascular system, which the bacteria subsequently colonize. Therefore, this study provides a new insight into the role of hydathodes in anti-bacterial resistance in plants and opens the door for research on tissue- and organ-specific resistance mechanisms.

In healthy plants, the innate immune system contributes to maintenance of microbiota homoeostasis, while disease can be associated with microbiome perturbation or dysbiosis, and enrichment of opportunistic plant pathogens like Xanthomonas. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter. Here we tested if protein export through the type-2 secretion system (T2SS) by Xanthomonas causes microbiome dysbiosis in Arabidopsis thaliana in immunocompromised plants. We found that Xanthomonas strains secrete a cocktail of plant cell wall-degrading enzymes that promote Xanthomonas growth during infection. Disease severity and leaf tissue degradation were increased in A. thaliana mutants lacking the NADPH oxidase RBOHD. Experiments with gnotobiotic plants, synthetic bacterial communities and wild-type or T2SS-mutant Xanthomonas revealed that virulence and leaf microbiome composition are controlled by the T2SS. Overall, a compromised immune system in plants can enrich opportunistic pathogens, which damage leaf tissues and ultimately cause microbiome dysbiosis by facilitating growth of specific commensal bacteria.

Dysbiosis is characterized by a perturbed microbiota associated with host disease. In both plants and animals, the innate immune system contributes to maintain microbiota homeostasis in healthy organisms, with NADPH oxidases playing a crucial role. In Arabidopsis thaliana , the absence of NADPH oxidase RBOHD can lead to an altered leaf microbiota, including an enrichment of opportunistic Xanthomonas pathogens. It is currently unclear whether the microbiota change occurs independently of the opportunistic pathogens or is caused by the latter, and which virulence factors of Xanthomonas are essential for its opportunistic lifestyle. Here, we found that the opportunistic Xanthomonas strains secrete a cocktail of cell wall degrading enzymes via the type-2 secretion system (T2SS) that degrade leaf tissue and promote Xanthomonas growth during plant infection. Both disease severity and leaf degradation activity were increased in rbohD compared to Col-0 plants, attesting to the opportunistic behaviour of the Xanthomonas strains on immune compromised plants. Using gnotobiotic plant experiments with a synthetic bacterial community of more than 100 commensal strains and drop-in of Xanthomonas wildtype or mutant strains revealed that T2SS-dependent virulence is required for plant disease and for the shift in microbiota composition. Overall, our data indicate that a single opportunistic pathogen can drive community shifts, here caused by tissue damage in leaves, creating an environment in which specific commensal bacteria can thrive.

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance, and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato (Pst) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival, and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT–PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature, and surface association strongly affect the expression of different polysaccharides under the control of the signalling protein genes ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides play a role in mediating leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection.

Prior to infection, phytopathogenic bacteria face a challenging environment on the plant surface, where they are exposed to nutrient starvation and abiotic stresses. Pathways enabling surface adhesion, stress tolerance and epiphytic survival are important for successful plant pathogenesis. Understanding the roles and regulation of these pathways is therefore crucial to fully understand bacterial plant infections. The phytopathogen Pseudomonas syringae pv. tomato ( Pst ) encodes multiple polysaccharides that are implicated in biofilm formation, stress survival and virulence in other microbes. To examine how these polysaccharides impact Pst epiphytic survival and pathogenesis, we analysed mutants in multiple polysaccharide loci to determine their intersecting contributions to epiphytic survival and infection. In parallel, we used qRT-PCR to analyse the regulation of each pathway. Pst polysaccharides are tightly coordinated by multiple environmental signals. Nutrient availability, temperature and surface association strongly affect the expression of different polysaccharides under the control of the signalling proteins ladS and cbrB and the second messenger cyclic-di-GMP. Furthermore, functionally redundant, combinatorial phenotypes were observed for several polysaccharides. Exopolysaccharides and WapQ-mediated lipopolysaccharide production are important for leaf adhesion, while α-glucan and alginate together confer desiccation tolerance. Our results suggest that polysaccharides play important roles in overcoming environmental challenges to Pst during plant infection. Highlight: Pseudomonas syringae uses the coordinated deployment of polysaccharides to address environmental challenges during plant colonization. Functional redundancy renders individual polysaccharides dispensable during plant infection, but their combined loss impedes pathogenicity.

Plants, like other multicellular lifeforms, are colonized by microorganisms. How plants respond to their microbiota is currently not well understood. We used a phylogenetically diverse set of 39 endogenous bacterial strains from Arabidopsis thaliana leaves to assess host transcriptional and metabolic adaptations to bacterial encounters. We identified a molecular response, which we termed the general non-self response (GNSR) that involves the expression of a core set of 24 genes. The GNSR genes are not only consistently induced by the presence of most strains, they also comprise the most differentially regulated genes across treatments and are predictive of a hierarchical transcriptional reprogramming beyond the GNSR. Using a complementary untargeted metabolomics approach we link the GNSR to the tryptophan-derived secondary metabolism, highlighting the importance of small molecules in plant–microbe interactions. We demonstrate that several of the GNSR genes are required for resistance against the bacterial pathogen Pseudomonas syringae. Our results suggest that the GNSR constitutes a defence adaptation strategy that is consistently elicited by diverse strains from various phyla, contributes to host protection and involves secondary metabolism.