The Role of ClpV in the Physiology and Pathogenicity of Xanthomonas citri subsp. citri Strain zlm1908

Abstract

Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium responsible for citrus canker, a significant threat to citrus crops. ClpV is a critical protein in the type VI secretion system (T6SS) as an ATPase involved in bacterial motility, adhesion, and pathogenesis to the host for some pathogenic bacteria. In order to investigate the function of clpV gene in Xcc, the clpV-deletion strain ΔclpV was constructed, its biological properties were evaluated, and the differences in gene expression levels between the wild-type strain and ΔclpV were analyzed by transcriptomics. The results exhibited significantly reduced biofilm formation, extracellular polysaccharide synthesis, and swarming motility in ΔclpV compared to the wild-type strain. Although the clpV-deletion did not significantly affect bacterial growth or pathogenicity in terms of disease symptoms on citrus leaves, the mutant showed increased sensitivity to environmental stresses (NaCl, SDS, and H₂O₂) and antibiotics (β-lactams and aminoglycosides). Transcriptome analysis revealed that clpV-deletion altered the expression of motility-related genes and the efflux pump gene mexH. Our findings underscore the importance of ClpV in maintaining biofilm integrity and suggest a multifaceted role in adaptive strategies of Xcc, positioning ClpV as a potential target for mitigating citrus canker disease.

Keywords: T6SS; Xanthomonas citri subsp. citri; clpV; pathogenicity; physiologic function.

Figure 1

Construction of ΔclpV mutant strain of X. citri. subsp. citri strain zlm1908. (A) The strategy of clpV gene was knocked out by allele exchange in Xcc. (B) The construction of the deletion strain was confirmed by PCR amplification. M: DL2000 marker; lane 1: Xcc wild type; lane 2: ΔclpV mutant strain; lane 3: 10 generations of ΔclpV; lane 4: 20 generations of ΔclpV; lane 5: 30 generations of ΔclpV; lane 6: 40 generations of ΔclpV; lane 7: 50 generations of ΔclpV.

Figure 2

Morphological characteristics of X. citri subsp. citri strain zlm1908 (A), ΔclpV (B), and C-clpV (C) (SEM, 25,000× magnification).

Figure 3

The biological characteristics of ΔclpV mutant compared with Xcc wild type. (A) The growth curve of Xcc zlm1908 and ΔclpV in LB culture medium for 60 h; (B) biofilm formation; (C) swarming motilities; (D) susceptibility to various stress conditions; (E) the production of EPS; (F) extracellular enzyme activity: (F-a) extracellular amylase, (F-b) extracellular amylase hydrolysis circle diameter/colony diameter, (F-c) β-mannanase, and (F-d) cellulase. ns: no significant difference (p > 0.05) The experimental results were averaged over the three replicates, and the error bars represent standard deviations (SD). * p < 0.05; ** p < 0.001; ns, not significant (Student’s t-test) (n = 3 independent experiments).

Figure 4

Disease symptoms of the leaves at 15 days after inoculated with an OD₆₀₀ of 0.2 in C. sinensis ‘hongjiang’. (a) pinprick inoculation (b) infiltration inoculation.

Figure 5

Comparison of the global transcriptomes of Xcc zlm1908 and ΔclpV by RNA-Seq. (A) A total of 76 genes were significantly differentially expressed in ΔclpV compared to Xcc zlm1908, with absolute fold change >1 and FDR < 0.05. GO Classification: A bar chart depicting the gene ontology (GO) classification of the 39 DEGs, categorized into three classes: “Cellular Component” (CC), “Molecular Function” (MF), and “Biological Process” (BP). (B) KEGG enrichment upregulated pathways. (C) KEGG enrichment downregulated pathways.

Figure 6

Transcription verification of RNA-seq in the Xcc zlm1908 and ΔclpV by qRT-PCR. The results are representative of three independent experiments. * p < 0.05; ** p < 0.001.