To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.
Huanglongbing (HLB) or citrus greening disease is a destructive disease of citrus worldwide, which is associated with Candidatus Liberibacter asiaticus. This phloem-limited fastidious pathogen is transmitted by the Asian citrus psyllid, Diaphorina citri, and appears to be an intracellular pathogen that maintains an intimate association with the psyllid or the plant throughout its life cycle. The molecular basis of the interaction of this pathogen with its hosts is not well understood. We hypothesized that, during infection, Ca. L. asiaticus differentially expresses the genes critical for its survival and/or pathogenicity in either host. To test this hypothesis, quantitative reverse transcription-polymerase chain reaction was performed to compare the gene expression of Ca. L. asiaticus in planta and in psyllid. Overall, 381 genes were analysed for their gene expression in planta and in psyllid. Among them, 182 genes were up-regulated in planta compared with in psyllid (P < 0.05), 16 genes were up-regulated in psyllid (P < 0.05) and 183 genes showed no statistically significant difference (P ≥ 0.05) in expression between in planta and in psyllid. Our study indicates that the expression of the Ca. L. asiaticus genes involved in transcriptional regulation, transport system, secretion system, flagella assembly, metabolic pathway and stress resistance are changed significantly in a host-specific manner to adapt to the distinct environments of plant and insect. To our knowledge, this is the first large-scale study to evaluate the differential expression of Ca. L. asiaticus genes in a plant host and its insect vector.
The phytotoxin coronatine (COR) is produced by various pathovars of Pseudomonas syringae, including P. syringae pv. tomato DC3000, which is pathogenic on crucifers and tomato, and P. syringae pv. glycinea PG4180, a soybean pathogen. The COR molecule contains two distinct components, coronafacic acid (CFA) and coronamic acid (CMA), which are intermediates in the COR biosynthetic pathway. In P. syringae pv. tomato DC3000, it is not clear whether corR, which encodes a response regulator, positively regulates CFA and CMA synthesis as it does in P. syringae pv. glycinea PG4180. In this study, a corR mutant of P. syringae pv. tomato DC3000 was constructed and was shown to be defective in the production of COR, CFA, and CMA. Furthermore, disease severity was greatly reduced in tomato plants inoculated with the corR mutant compared with wild-type P. syringae pv. tomato DC3000. We also showed that a mutation in hrpL, which encodes an alternate RNA polymerase sigma factor (sigmaL) required for the expression of genes encoding components of the type III secretion system, abrogated production of COR in P. syringae pv. tomato DC3000. The presence of a potential hrp box, the recognition site for sigmaL, upstream of corR suggested that corR might be regulated by hrpL. This was confirmed in reverse-transcription polymerase chain reaction experiments showing that the upstream effector gene holPtoAA, which was associated with the hrp box, was cotranscribed with corR. Furthermore, studies also were conducted to investigate whether mutations in corR had effects on the expression of hrpL. The corR mutant of P. syringae pv. tomato DC3000 showed both a reduction and delay in the expression of hrpL and was impaired in its ability to elicit a hypersensitive response on Nicotiana benthamiana. A putative CorR-binding site was identified upstream of hrpL, and gel shift studies confirmed the binding of CorR to this region. These results indicate that corR directly impacts the expression of the hrp regulon in P. syringae.
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