New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library

Abstract

Background: Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis.

Results: Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands.

Conclusion: The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.

Figure 1

Southern blot analysis shows transposon isertion. X-ray film image after exposure to DNA of Xanthomonas citri subsp. citri strain 306 isolated mutant clones, previously cleaved with Eco RI and hybridized with the sequence of the transposon Tn5 labeled with the AlkPhos Direct RPN 3680 kit (Amersham Biosciences). Mutants with a double insert are marked with an asterisk.

Figure 2

Xcc growth curves. Growth curves of 16 Xanthomonas citri subsp. citri mutants and wild type (Xcc strain 306) in vitro (left) and in citrus leaves (right).

Figure 3

Nucleic acid hybridization using labeled cDNA probes. Nucleic acid hybridization using labeled cDNA probe to 11 Xanthomonas citri subsp. citri strain 306 (Xcc) genes identified as important for pathogenicity through random mutagenesis. Panel A = gene expression of ORFs when Xcc was multiplied in culture medium. Panel B = gene expression of ORFs when Xcc was multiplied in citrus leaves for 3 days. C1–C4 = controls (5 ng, 20 ng, 80 ng and 320 ng, respectively).

Figure 4

Schematic representation of a suggested DSF signaling model including XAC3673. Schematic representation of a suggested DSF signaling model including XAC3673. At a low cell density, the DSF sensor RpfC forms a complex with the DSF synthase RpfF, which prevents the effective synthesis of the DSF signal. At a high cell density, accumulated extracellular DSF signal interacts with RpfC, which undergoes autophosphorylation and facilitates release of RpfF and phosphorelay from the sensor to its response regulator RpfG. The event boosts DSF biosynthesis and induces the expression of the EPS and extracelular enzymes. In either, low or high cell density, there may be other stimuli (signals), in the extracellular environment from the host or the environment, regardless of the bacterial cellular concentration. The synthesis of Xcc virulence factors only start after the perception of such signals. XAC3673, through a phosphorylation cascade, relays this information to RpfG or to another protein downstream (arrows with yellow lines). A mutation in XAC3673 prevents the transduction of signals from the environment or host, and thus, the virulence factors are not produced, even in the presence of all functional rpf genes and with a high cell concentration. The solid arrow indicates signal flow or signal generation and the dashed arrow indicates basal signal generation or no signal flow. OM = outer membrane; IM = inner membrane.

Figure 5

Xcc genome exclusive regions. Determination of possible Xcc exclusive regions on the basis of analysis of mutant (upstream and downstream) flanking regions. Five regions were found (1–5), three very close to each other (3–5). Notice that all regions have a GC content and codon bias sequence that differs from the rest of the genome profile. Details of each region are shown. Note that regions 1 and 4 are Xcc exclusive regions.