Clavibacter michiganensis subsp.
michiganensis is a Gram-positive bacterium that causes wilting and cankers, leading to severe economic losses in commercial tomato production worldwide. The disease is transmitted from infected seeds to seedlings and mechanically from plant to plant during seedling production, grafting, pruning, and harvesting. Because of the lack of tools for genetic manipulation, very little is known regarding the mechanisms of seed and seedling infection and movement of
C. michiganensis subsp.
michiganensis in grafted plants, two focal points for application of bacterial canker control measures in tomato. To facilitate studies on the
C. michiganensis subsp.
michiganensis movement in tomato seed and grafted plants, we isolated a bioluminescent
C. michiganensis subsp.
michiganensis strain using the modified Tn
1409 containing a promoterless
lux reporter. A total of 19 bioluminescent
C. michiganensis subsp.
michiganensis mutants were obtained. All mutants tested induced a hypersensitive response in
Mirabilis jalapa and caused wilting of tomato plants. Real-time colonization studies of germinating seeds using a virulent, stable, constitutively bioluminescent strain, BL-Cmm17, showed that
C. michiganensis subsp.
michiganensis aggregated on hypocotyls and cotyledons at an early stage of germination. In grafted seedlings in which either the rootstock or scion was exposed to BL-Cmm17 via a contaminated grafting knife, bacteria were translocated in both directions from the graft union at higher inoculum doses. These results emphasize the use of bioluminescent
C. michiganensis subsp.
michiganensis to help better elucidate the
C. michiganensis subsp.
michiganensis-tomato plant interactions. Further, we demonstrated the broader applicability of this tool by successful transformation of
C. michiganensis subsp.
nebraskensis with Tn
1409::
lux. Thus, our approach would be highly useful to understand the pathogenesis of diseases caused by other subspecies of the agriculturally important
C. michiganensis.Clavibacter michiganensis subsp.
michiganensis is a Gram-positive, aerobic bacterium that belongs to a group of plant-pathogenic actinomycetes (
37). Infections by
C. michiganensis subsp.
michiganensis cause bacterial canker and wilt in tomato, which is considered one of the most destructive and economically significant diseases of this crop. Severe epidemics can cause up to 80% yield loss, mainly due to wilting and death of plants and lesions on fruit. Bacterial canker was first discovered in Michigan greenhouses in 1909 and has now been reported to occur in most tomato production areas around the world (
11,
40).Plant wounds facilitate but are not required for infection by
C. michiganensis subsp.
michiganensis, which invades the xylem vessels and causes vascular disease with high titers (10
9 bacteria/g of plant tissue) (
2,
29), impairing water transport and leading to plant wilting, canker stem lesions, and death (
17,
23). Alternatively, asymptomatic infections can be induced by
C. michiganensis subsp.
michiganensis during late stages of plant development, resulting in the production of contaminated seeds, a major source of outbreaks of
C. michiganensis subsp.
michiganensis infections in tomato production (
13,
34). Traditional bacterial-disease management measures, such as applications of antibiotics and copper bactericides, have not been successful against this disease, and canker-resistant tomato cultivars are not available. As a result,
C. michiganensis subsp.
michiganensis has been included under international quarantine regulation (
10,
11). Consequently, seed testing and maintaining pathogen-free seeds and transplants is currently the most appropriate approach to minimize the spread of disease (
23). However, even a low
C. michiganensis subsp.
michiganensis transmission rate (0.01%) from seed to seedling can cause a disease epidemic under favorable conditions (
5). Due to overcrowding of seedlings during transplant production, the pathogen can easily spread through splashing of irrigation water and leaf contact. Despite its apparent significance in
C. michiganensis subsp.
michiganensis epidemiology, the mechanism of seed-to-seedling transmission of
C. michiganensis subsp.
michiganensis is not well understood.Another critical point for disease spread is the grafting process, which is now a common practice for the majority of plants used in production greenhouses. Desirable tomato cultivars (scions) are grafted onto rootstocks that provide greater vigor, longevity, or, in some cases, disease resistance (
26). Grafting requires cutting both rootstock and scion, providing a quick way for
C. michiganensis subsp.
michiganensis to spread from plant to plant. However, grafting is a relatively recent innovation in tomato production, and little is known about how grafting affects the dynamics of
C. michiganensis subsp.
michiganensis infection. Developing adequate control measures for
C. michiganensis subsp.
michiganensis is complicated by the complexity of genetic manipulation of Gram-positive bacteria, which impairs analysis and characterization of pathogenesis mechanisms (
23). Consequently, there is a need to develop molecular techniques that would allow a better understanding of
C. michiganensis subsp.
michiganensis infections.One method of interest is using engineered bioluminescent bacteria to monitor plant-pathogen interactions in real time. By exploiting natural light-emitting reactions that are encoded by the
luxCDABE genes, bioluminescent bacteria have been used to assess gene expression and to monitor the internalization and distribution of bacteria in hosts (
3,
6,
7,
8,
9,
12,
15,
24,
31,
35,
36). In particular, bioluminescent phytopathogenic
Xanthomonas campestris pathovars and
Pseudomonas spp. have been used to track bacterial movement and distribution in host plants (
7,
8,
15,
31,
36), as well as to assess host susceptibility quantitatively (
15). Likewise, the
lux genes have also been transferred to beneficial bacteria, such as
Rhizobium leguminosarum and
Pseudomonas spp. to visualize colonization patterns in rhizospheres (
3,
9).The genes that carry the function of light emission are
luxAB, which express luciferase enzymes that catalyze the bioluminescent reaction, while
luxCDE encode the enzymes required for biosynthesis of a fatty aldehyde substrate necessary for the reaction (
28,
39). Bioluminescence involves an intracellular oxidation of the reduced form of flavin mononucleotide and the fatty aldehyde by luciferase in the presence of molecular oxygen; therefore, bacterial bioluminescence also requires oxygen, a source of energy (
38). Cells that express the
lux operon spontaneously emit photons that can be captured by a sensitive charge-coupled-device (CCD) camera, enabling imaging and visualization of bacterial cells (
22). Luciferase activity depends on the metabolic integrity of the cell, while the number of photons emitted correlates with the biomass of living bacteria (
12,
31). Furthermore, since the half-life of luciferase binding to its substrate is several seconds (
28), captured light events reflect processes in real time and are not artifacts of accumulated signals. Consequently, live imaging of bioluminescence provides a sensitive means of visualizing bacterial colonization and invasion of hosts and allows real-time representation and examination of pathogen-plant interactions (
24,
36).Very little information is available about the mechanisms of
C. michiganensis subsp.
michiganensis pathogenesis and its colonization of seeds and subsequent transmission to seedlings. This is largely attributable to a lack of tools and difficulties in genetically manipulating this Gram-positive bacterium (
30). However, recent development of an insertion sequence element IS
1409 (Tn
1409)-based efficient transposon mutagenesis system for
C. michiganensis subsp.
michiganensis has increased our knowledge of the pathogenesis of tomato canker (
16,
25). To better understand the dynamics of seed-to-seedling transmission of
C. michiganensis subsp.
michiganensis, as well as movement of
C. michiganensis subsp.
michiganensis in grafted plants, we constructed a bioluminescent
C. michiganensis subsp.
michiganensis strain using the Tn
1409 transposon mutagenesis system. Our results demonstrated the utility of using a bioluminescent
C. michiganensis subsp.
michiganensis strain as a novel approach to elucidate the interaction of plants with this economically important pathogen.
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