A Genetic Toolkit for Investigating Clavibacter Species: Markerless Deletion, Permissive Site Identification, and an Integrative Plasmid

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advances in these techniques in gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens that cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen Clavibacter michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination-based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and was confirmed using whole-genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a nonreplicating integrative plasmid that enables recombination into a permissive genomic location. Expression of enhanced green fluorescent protein by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advances for investigating gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

A genetic toolkit for investigating Clavibacter: markerless deletion, permissive site identification and an integrative plasmid

The development of knockout mutants and expression variants are critical for understanding genotype-phenotype relationships. However, advancements of these techniques in Gram-positive actinobacteria have stagnated over the last decade. Actinobacteria in the Clavibacter genus are composed of diverse crop pathogens which cause a variety of wilt and cankering diseases. Here, we present a suite of tools for genetic manipulation in the tomato pathogen C. michiganensis including a markerless deletion system, an integrative plasmid, and an R package for identification of permissive sites for plasmid integration. The vector pSelAct-KO is a recombination based, markerless knockout system that uses dual selection to engineer seamless deletions of a region of interest, providing opportunities for repeated higher-order genetic knockouts. The efficacy of pSelAct-KO was demonstrated in C. michiganensis and confirmed using whole genome sequencing. We developed permissR, an R package to identify permissive sites for chromosomal integration, which can be used in conjunction with pSelAct-Express, a non-replicating integrative plasmid that enables recombination into a permissive genomic location. Expression of eGFP by pSelAct-Express was verified in two candidate permissive regions predicted by permissR in C. michiganensis. These molecular tools are essential advancements for investigating Gram-positive actinobacteria, particularly for important pathogens in the Clavibacter genus.

A diverse member of the fungal Avr4 effector family interacts with de-esterified pectin in plant cell walls to disrupt their integrity

Effectors are small, secreted proteins that promote pathogen virulence. Although key to microbial infections, unlocking the intrinsic function of effectors remains a challenge. We have previously shown that members of the fungal Avr4 effector family use a carbohydrate-binding module of family 14 (CBM14) to bind chitin in fungal cell walls and protect them from host chitinases during infection. Here, we show that gene duplication in the Avr4 family produced an Avr4-2 paralog with a previously unknown effector function. Specifically, we functionally characterize PfAvr4-2, a paralog of PfAvr4 in the tomato pathogen Pseudocercospora fuligena, and show that although it contains a CBM14 domain, it does not bind chitin or protect fungi against chitinases. Instead, PfAvr4-2 interacts with highly de-esterified pectin in the plant’s middle lamellae or primary cell walls and interferes with Ca2+-mediated cross-linking at cell-cell junction zones, thus loosening the plant cell wall structure and synergizing the activity of pathogen secreted endo-polygalacturonases.

Potassium Phosphite Modulated the Soil Microbiome and Enriched the Antagonistic Bacteria Streptomyces Coelicoflavus and Paenibacillus Favisporus to Inhibit the Tomato Pathogen Ralstonia Solanacearum Synergistically

Background: Application of certain agricultural chemicals could modulate the soil microbiome and induce potential antagonistic microbes. However, the specific selective effects of agricultural chemicals on soil bacterial functions and their co-occurrences are not well understood, and no studies have verified that the enriched potential antagonistic microbes could enhance the antagonistic functions of the soil microbiome.Results: Here, the effects of potassium phosphite (KP), an environment-friendly agricultural chemical, on the soil bacterial composition, co-occurrences and antagonistic functions were determined, and the potential antagonistic bacteria against the tomato bacterial wilt pathogen Ralstonia solanacearum were isolated to test their functions and associations among these strains. Our results showed that application of KP enriched Bacillus, Paenibacillus and Streptomyces. The positive links among the OTUs belonging to these genera were increased, and positive associations between these OTUs and predicted genes related to antagonistic substance production were revealed. Two strains, Streptomyces coelicoflavus F13 and Paenibacillus favisporus Y7, were isolated, and they inhibited the growth of R. solanacearum. Genomic sequencing showed that both strains harboured streptomycin synthetic genes, and P. favisporus Y7 also contained surfactin synthetic gene cluster. Synergistic inhibition of R. solanacearum growth by P. favisporus Y7 and S. coelicoflavus F13 was observed in soil. Genome-scale metabolic modelling showed that dextrin and lactic acid were potential cross-feeding metabolites. In addition, the KP-modulated soil microbiome could suppress R. solanacearum growth. Conclusions: Our results highlight that a KP-modulated soil microbiome has considerable potential for biocontrol and indicate a new mechanism for the inhibition of R. solanacearum by KP-enriched soil bacteria.

A homologue of the mammalian tumour suppressor protein PTEN is a functional lipid phosphatase and required for chemotaxis in filamentous fungi

SummaryPhosphoinositides (PI) are essential components of eukaryotic membranes and function in a large number of signalling processes. While lipid second messengers are well studied in mammals and yeast, their role in filamentous fungi is poorly understood. We used fluorescent PI-binding molecular probes to localise the phosphorylated phosphatidylinositol species PI[3]P, PI[3,5]P2, PI[4]P and PI[4,5]P2 in hyphae of the endophyte Epichloë festucae in axenic culture and during interaction with its grass host Lolium perenne. We also analysed the roles of the phosphatidylinositol-4-phosphate 5-kinase MssD and the predicted phosphatidylinositol-3,4,5-triphosphate 3-phosphatase TepA, a homologue of the mammalian tumour suppressor protein PTEN. Deletion of tepA in E. festucae and in the root-infecting tomato pathogen Fusarium oxysporum had no impact on growth in culture or the host interaction phenotype. However, this mutation did uncover the presence of PI[3,4,5]P3 in septa of E. festucae and showed that TepA is required for chemotropism in F. oxysporum. The identification of PI[3,4,5]P3 in septa of ΔtepA strains suggests that filamentous fungi are able to generate PI[3,4,5]P3 using an alternative biosynthetic pathway and that fungal PTEN homologues are functional lipid phosphatases. The F. oxysporum chemotropism defect demonstrates a conserved role of PTEN homologues in chemotaxis across protists, fungi and mammals.

Non-Destructive Early Detection and Quantitative Severity Stage Classification of Tomato Chlorosis Virus (ToCV) Infection in Young Tomato Plants Using Vis–NIR Spectroscopy

Tomato chlorosis virus (ToCV) is a serious, emerging tomato pathogen that has a significant impact on the quality and quantity of tomato production worldwide. Detecting ToCV via means of spectral measurements in an early pre-symptomatic stage offers an alternative to the existing laboratory methods, leading to better disease management in the field. In this study, leaf spectra from healthy and diseased leaves were measured with a spectrometer. The diseased leaves were subjected to RT-qPCR for the detection and quantification of the titer of ToCV. Neighborhood component analysis (NCA) algorithm was employed for the feature selection of the effective wavelengths and the most important vegetation indices out of the 24 that were tested. Two machine learning methods, namely XY-fusion network (XY-F) and multilayer perceptron with automated relevance determination (MLP–ARD), were employed for the estimation of the disease existence and viral load in the tomato leaves. The results showed that before outlier elimination, the MLP–ARD classifier generally outperformed the XY-F network with an overall accuracy of 92.1% against 88.3% for the XY-F. Outlier elimination contributed to the performance of the classifiers as the overall accuracy for both XY-F and MLP–ARD reached 100%.

The Bacterial Effector AvrPto Targets the Regulatory Coreceptor SOBIR1 and Suppresses Defense Signaling Mediated by the Receptor-Like Protein Cf-4

Receptor-like proteins (RLPs) and receptor-like kinases (RLKs) are cell-surface receptors that are essential for detecting invading pathogens and subsequent activation of plant defense responses. RLPs lack a cytoplasmic kinase domain to trigger downstream signaling leading to host resistance. The RLK SOBIR1 constitutively interacts with the tomato RLP Cf-4, thereby providing Cf-4 with a kinase domain. SOBIR1 is required for Cf-4–mediated resistance to strains of the fungal tomato pathogen Cladosporium fulvum that secrete the effector Avr4. Upon perception of this effector by the Cf-4/SOBIR1 complex, the central regulatory RLK SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3a (SERK3a) is recruited to the complex and defense signaling is triggered. SOBIR1 is also required for RLP-mediated resistance to bacterial, fungal ,and oomycete pathogens, and we hypothesized that SOBIR1 is targeted by effectors of such pathogens to suppress host defense responses. In this study, we show that Pseudomonas syringae pv. tomato DC3000 effector AvrPto interacts with Arabidopsis SOBIR1 and its orthologs of tomato and Nicotiana benthamiana, independent of SOBIR1 kinase activity. Interestingly, AvrPto suppresses Arabidopsis SOBIR1-induced cell death in N. benthamiana. Furthermore, AvrPto compromises Avr4-triggered cell death in Cf-4-transgenic N. benthamiana, without affecting Cf-4/SOBIR1/SERK3a complex formation. Our study shows that the RLP coreceptor SOBIR1 is targeted by a bacterial effector, which results in compromised defense responses.

Genome-Wide Analysis of Small Secreted Cysteine-Rich Proteins Identifies Candidate Effector Proteins Potentially Involved in Fusarium graminearum−Wheat Interactions

Pathogen-derived, small secreted cysteine-rich proteins (SSCPs) are known to be a common source of fungal effectors that trigger resistance or susceptibility in specific host plants. This group of proteins has not been well studied in Fusarium graminearum, the primary cause of Fusarium head blight (FHB), a devastating disease of wheat. We report here a comprehensive analysis of SSCPs encoded in the genome of this fungus and selection of candidate effector proteins through proteomics and sequence/transcriptional analyses. A total of 190 SSCPs were identified in the genome of F. graminearum (isolate PH-1) based on the presence of N-terminal signal peptide sequences, size (≤200 amino acids), and cysteine content (≥2%) of the mature proteins. Twenty-five (approximately 13%) SSCPs were confirmed to be true extracellular proteins by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis of a minimal medium-based in vitro secretome. Sequence analysis suggested that 17 SSCPs harbor conserved functional domains, including two homologous to Ecp2, a known effector produced by the tomato pathogen Cladosporium fulvum. Transcriptional analysis revealed that at least 34 SSCPs (including 23 detected in the in vitro secretome) are expressed in infected wheat heads; about half are up-regulated with expression patterns correlating with the development of FHB. This work provides a solid candidate list for SSCP-derived effectors that may play roles in mediating F. graminearum−wheat interactions. The in vitro secretome-based method presented here also may be applicable for identifying candidate effectors in other ascomycete pathogens of crop plants.