a-toxin-based-counter-selection-system-for-markerless-gene-deletion

A Toxin-Based Counter-Selection System for Markerless Gene Deletion and High-Density Tn5 Transposon Mutagenesis in <em>Pectobacterium brasiliense</em>

Pectobacterium brasiliense is a highly pathogenic bacterium responsible for potato tuber soft rot, blackleg, and aerial stem rot. Elucidation of its ...

Agricultural Research Organization-Volcani Institute

Bacterial wilt and canker caused by Clavibacter michiganensis (Cm) inflict considerable damage in tomato-growing regions around the world. Cm has a narrow host range and can cause disease in tomato but not in many eggplant varieties. The pathogenicity of Cm is dependent on secreted serine proteases, encoded by the chp/tomA pathogenicity island (PI), and the pCM2 plasmid. Screening combinations of PI deletion mutants and plasmid-cured strains found that Cm-mediated hypersensitive response (HR) in the Cm-resistant eggplant variety Black Queen is dependent on the chp/tomA PI. Singular reintroduction of PI-encoded serine proteases into Cm identified that the HR is elicited by the protease ChpG. Eggplant leaves infiltrated with a chpG marker exchange mutant (CmΩchpG) did not display an HR, and infiltration of purified ChpG protein elicited immune responses in eggplant but not in Cm-susceptible tomato. Virulence assays found that while wild-type Cm and the CmΩchpG complemented strain were nonpathogenic on eggplant, CmΩchpG caused wilt and canker symptoms. Additionally, bacterial populations in CmΩchpG-inoculated eggplant stem tissues were c.1000-fold higher than wild-type and CmΩchpG-complemented Cm strains. Pathogenicity tests conducted in multiple Cm-resistance eggplant varieties demonstrated that immunity to Cm is dependent on ChpG in all tested varieties, indicating that ChpG-recognition is conserved in eggplant. ChpG-mediated avirulence interactions were disabled by alanine substitution of serine231 of the serine protease catalytic triad, suggesting that protease activity is required for immune recognition of ChpG. Our study identified ChpG as a novel avirulence protein that is recognized in resistant eggplant varieties and restricts the host range of Cm.

Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties.

Plant pathogenic bacteria often have a narrow host range, which can vary among different isolates within a population. Here, we investigated the host range of the tomato pathogen Clavibacter michiganensis (Cm). We determined the genome sequences of 40 tomato Cm isolates and screened them for pathogenicity on tomato and eggplant. Our screen revealed that out of the tested isolates, five were unable to cause disease on any of the hosts, 33 were exclusively pathogenic on tomato, and two were capable of infecting both tomato and eggplant. Through comparative genomic analyses, we identified that the five non-pathogenic isolates lacked the chp/tomA pathogenicity island, which has previously been associated with virulence in tomato. In addition, we found that the two eggplant-pathogenic isolates encode a unique allelic variant of the putative serine hydrolase chpG (chpGC), an effector that is recognized in eggplant. Introduction of chpGC into a chpG inactivation mutant in the eggplant-non-pathogenic strain Cm101, failed to complement the mutant, which retained its ability to cause disease in eggplant and failed to elicit hypersensitive response (HR). Conversely, introduction of the chpG variant from Cm101 into an eggplant pathogenic Cm isolate (C48), eliminated its pathogenicity on eggplant, and enabled C48 to elicit HR. Our study demonstrates that allelic variation in the chpG effector gene is a key determinant of host range plasticity within Cm populations.

Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range.

 species are specialized plant pathogens, often exhibiting a narrow host range. They rely on the translocation of effector proteins through the type III secretion system to colonize their respective hosts. The effector arsenal varies among  spp., typically displaying species-specific compositions. This species-specific effector composition, collectively termed the effectorome, is thought to influence host specialization. We determined the plant host-derived effectoromes of more than 300 deposited genomes of  species associated with either  or  hosts. Comparative analyses revealed clear species-specific effectorome signatures. However,  or  host-associated effectorome signatures were not detected. Nevertheless, host biases in the presence or absence of specific effector classes were observed. To assess whether host-associated effector absence results from selective pressures, we introduced effectors unique to  pathogens to  pv.  and effectors unique to  pathogens to  pv.  (Xeue) and evaluated if these introductions hindered virulence on their respective hosts. Introducing the effector XopI into  pv.  reduced virulence on white cabbage leaves without affecting localized or systemic colonization. Introducing the XopAC or XopJ5 effectors into Xeue reduced virulence and colonization on tomato but not on pepper. Additionally, XopAC and XopJ5 induced a hypersensitive response on tomato leaves when delivered by Xeue or through -mediated transient expression, confirming recognition in tomato. This study demonstrates the role of host-derived selection in establishing species-specific effectoromes, identifying XopAC and XopJ5 as recognized effectors in tomato.

Host-Driven Selection, Revealed by Comparative Analysis of  Type III Secretion Effectoromes, Unveils Novel Recognized Effectors.

 pv.  () induces tumor-like galls in beet and gypsophila, a process mediated by the secretion of effector proteins via 's type III secretion system (T3SS). This study delves into the cellular architecture and transcriptional profile of -mediated galls, comparing host responses with wild-type  and a T3SS-inactive mutant, . Morphological analysis using scanning electron microscopy and cross-sectional visualization of infected beet leaf tissues revealed that -induced gall-like structures are linked to cell hyperplasia and tissue ruptures, contingent on T3SS activity. Comparative transcriptome analysis of wild-type  and  -infected beet leaves at 12 and 48 h unveiled significant transcriptional reprogramming, with nearly 2,000 differentially expressed genes at 48 h postinoculation. Enrichment analyses identified the upregulation of pathways related to signal transduction, defense, carbohydrate metabolism, and cell wall modulation in wild-type -infected leaves compared with controls. Particularly notable was the significant upregulation of numerous genes associated with cell wall loosening by wild-type , suggesting that an initial rearrangement of cell wall architecture facilitates gall formation. Furthermore, transcriptome analysis demonstrated that wild-type  suppresses the expression of the betalain biosynthetic gene , leading to reduced betalain accumulation in infected tissues compared with the mutant strain. These findings offer fresh insights into the transcriptional and physiological manipulation of host tissue during the early stages of -induced gall formation.

Comparative Transcriptomic Profile Reveals Candidate Genes Manipulated by Type III Effectors of  pv.  Leading to Gall Formation in Beet.

The plant pathogenic bacterium Clavibacter michiganensis (Cm) is a systemic vascular pathogen that colonizes both xylem vessels and the intracellular apoplast during different stages of infection. To identify traits and loci associated with adaptation to these distinct host microenvironments, we conducted tissue-specific experimental evolution. Twenty independent Cm lineages were repeatedly passaged in either tomato stems or leaves to promote adaptation to vascular or apoplastic lifestyles, respectively. After fifteen passages, adapted clones were characterized for virulence and virulence-related traits. These characterizations demonstrated clear differential associations of virulence-associated traits with the adapted tissue. The majority of vascular-adapted clones displayed enhanced surface attachment, reduced cellulase activity, reduced exopolysaccharide (EPS) production, and attenuated virulence on tomato compared to the parent clone. In contrast, apoplast-adapted clones displayed reduced biofilm formation and enhanced EPS production and retained their virulence on tomato. Whole-genome sequencing of all adapted clones revealed candidate loci linked to tissue adaptation. Six of ten vascular-adapted clones carried two independent mutations in CMM_1284, a putative HipB/XRE-type transcriptional regulator. A CMM_1284 marker exchange mutant displayed phenotypes similar to vascular-adapted clones, suggesting a role for this regulator in vascular colonization. Together, these findings highlight the role of phenotypic plasticity in tissue adaptation of plant pathogens, showing that tissue-specific adaptation involves modulation of surface attachment, EPS production, and cell wall-degrading enzymes and suggest a trade-off between vascular persistence, supported by strong surface attachment, and systemic virulence, which depends on bacterial dispersal and migration.

Article	Year
Immune recognition of the secreted serine protease ChpG restricts the host range of Clavibacter michiganensis from eggplant varieties. Molecular plant pathology\| PubMed ID: 35441490	2022
Allelic variations in the chpG effector gene within Clavibacter michiganensis populations determine pathogen host range. PLoS pathogens\| PubMed ID: 39028765	2024
Host-Driven Selection, Revealed by Comparative Analysis of Type III Secretion Effectoromes, Unveils Novel Recognized Effectors. Phytopathology\| PubMed ID: 39133938	2024
Comparative Transcriptomic Profile Reveals Candidate Genes Manipulated by Type III Effectors of pv. Leading to Gall Formation in Beet. Phytopathology\| PubMed ID: 41213162	2026
Tissue-Specific Experimental Evolution Reveals Adaptive Trade-Offs in the Plant Vascular Pathogen Clavibacter michiganensis. The ISME journal\| PubMed ID: 42096544	2026

Doron Teper

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