Biology subjects

Kushwah, S.

Publications and source records attributed to Kushwah, S..

5 recordsLinked to original sources

bioRxiv2026

Mutation in Ralstonia pseudosolanacearum F1C1 cytochrome p450 homologue impairs virulence through root but not through shoot of tomato plants

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

pathology↗

bioRxiv2026

Morphology of petiole bending, senescence, epinasty, along with necrotic scarring in tomato leaves infiltrated with virulent Ralstonia pseudosolanacearum

Ralstonia pseudosolanacearum (Rps) belongs to the Ralstonia solanacearum species complex (RSSC). It is a vascular pathogen that causes lethal bacterial wilt disease in many plants, including tomato and eggplant. In this study, we infiltrated tomato leaves with the phytopathogenic bacterium at 109 CFU/mL and observed the development of necrotic scars in the infiltrated area at 48 hours post-infiltration. Interestingly, this response was followed by petiole bending toward the ground of the compound leaf. This was followed by the gradual senescence of the infiltrated leaflet only. In addition, the terminal leaflet infiltrated with the pathogen exhibited epinasty. None of the above symptoms were observed in leaves infiltrated with the known virulent deficient hrpB::{Omega} mutant. Surprisingly, all of the above symptoms were observed in leaves infiltrated with another well-known virulence-deficient mutant phcA::{Omega}. It indicated that the necrotic lesion caused in tomato leaves was hrp-dependent. Infiltration in eggplant leaves caused necrotic scarring and leaf senescence, which were relatively delayed. Necrotic scarring without petiole bending or senescence in tomato leaves was also observed due to infiltration of Pseudomonas aeruginosa SPT08, a tomato endophyte having plant growth promotion activity. The patho-phenotypes such as petiole bending, epinasty, and senescence observed in the case of tomato in this study were not reported earlier. We believe these phenotypes produced in tomato after leaf infiltration may be useful to study the virulence of this pathogen.

pathology↗

bioRxiv2025

Targeting Metabolic Vulnerabilities: Valinomycin Augments the Potency of Bioenergetic Inhibitors in Combatting Drug-Resistant and Dormant Mycobacterium tuberculosis

Tuberculosis (TB) treatment is hampered by monotherapy limitations and phenotypic drug tolerance, citing the need for effective drug combinations. The mycobacterial electron transport chain (ETC), crucial for oxidative phosphorylation and ATP production in dormant Mycobacterium tuberculosis (Mtb), is a key target. This study investigates combining established bioenergetic inhibitors-bedaquiline (BDQ), telacebec (Q203), and clofazimine (CFZ) with the potassium ionophore valinomycin, which disrupts the proton motive force (pmf). We demonstrated that valinomycin significantly potentiated the anti-TB activity of these inhibitors against both replicating and nutrient-starved non-replicating Mtb. Checkerboard assays revealed synergistic activity with BDQ and additive effects with Q203 and CFZ, correlated with a two to three-fold reduction in ATP IC50 values. Critically, valinomycin converted the bacteriostatic activity of the inhibitors at sub-MIC concentration into bactericidal killing in time-dependent killing assay, achieving sterilization. This lethal synergy for the combinations was also observed in a THP-1 macrophage intracellular model for TB. Respiration assays confirmed that the combinations collectively halted oxygen consumption. We conclude that concurrently targeting specific ETC on Mtbs bioenergetics. This strategy, particularly the BDQ/valinomycin synergy, represents a promising cornerstone for developing novel sterilizing regimens to shorten TB therapy and overcoming drug tolerance.

microbiology↗

bioRxiv2021

The phosphate starvation response recruits the TOR pathway to regulate growth in Arabidopsis cell cultures

AO_SCPLOWBSTRACTC_SCPLOWIn eukaryotes, TOR (Target Of Rapamycin) is a conserved regulator of growth that integrates both endogenous and exogenous signals. These signals include the internal nutritional status, and in plants, TOR has been shown to be regulated by carbon, nitrogen and sulfur availability. In this study, we show that in Arabidopsis the TOR pathway also integrates phosphorus availability to actively modulate the cell cycle, which in turn regulates the intracellular content of amino acids and organic acids. We observed a substantial overlap between the phenotypic, metabolic and transcriptomic responses of TOR inactivation and phosphorus starvation in Arabidopsis cell culture. Although phosphorus availability modulates TOR activity, changes in the levels of TOR activity do not alter the expression of marker genes for phosphorus status. These data prompted us to place the sensing of phosphorus availability upstream of the modulation of TOR activity which, in turn, regulates the cell cycle and primary metabolism to adjust plant growth in plants.

plant biology↗

bioRxiv2019

Arabidopsis XTH4 and XTH9 contribute to wood cell expansion and secondary wall formation

In dicotyledons, xyloglucan is the major hemicellulose of primary walls affecting the load-bearing framework with participation of XTH enzymes. We used loss- and gain-of function approaches to study functions of abundant cambial region expressed XTH4 and XTH9 in secondary growth. In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. In addition, they stimulated secondary wall thickening, but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition, indicating overall increase in secondary versus primary walls in the mutants, indicative of higher xylem production compared to wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers was increased in xth9 and reduced in xth4, whereas the double mutant xth4x9 displayed intermediate number of layers. These changes correlated with certain Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers where neither XTH4 nor XTH9 were expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and in cell wall integrity sensing, including THE1 and WAK2, were highly induced in the mutants, indicating that deficiency in XTH4 and XTH9 triggers cell wall integrity signaling, which, we propose, stimulates the xylem cell production and modulates secondary wall thickening. Prominent effects of XTH4 and XTH9 on secondary xylem support the hypothesis that altered xyloglucan can affect wood properties both directly and via cell wall integrity sensing. SIGNIFICANCE STATEMENTXyloglucan is a ubiquitous component of primary cell walls in all land plants but has not been so far reported in secondary walls. It is metabolized in muro by cell wall-residing enzymes - xyloglucan endotransglycosylases/hydrolases (XTHs), which are reportedly abundant in vascular tissues, but their role in these tissues is unclear. Here we report that two vascular expressed enzymes in Arabidopsis, XTH4 and XTH9 contribute to the secondary xylem cell radial expansion and intrusive elongation in secondary vascular tissues. Unexpectedly, deficiency in their activities highly affect chemistry and ultrastructure of secondary cell walls by non-cell autonomous mechanisms, including transcriptional induction of secondary wall-related biosynthetic genes and cell wall integrity sensors. These results link xyloglucan metabolism with cell wall integrity pathways, shedding new light on previous reports about prominent effects of xyloglucan metabolism on secondary walls. One sentence summaryXTH4 and XTH9 positively regulate xylem cell expansion and fiber intrusive tip growth, and their deficiency alters secondary wall formation via cell wall integrity sensing mechanisms.

developmental biology↗