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Lemon tree fruit lesions of the vascular wilt fungus *Plasmopara viticola* exhibit a typical lesion with dead cells and no living cells ([@b14-ppj-34-207]). In an early infection, fungal hyphae colonize cells in a distal part of the vascular bundle, then migrate toward the stele and other vessels ([@b9-ppj-34-207], [@b10-ppj-34-207]). In general, the infection process is divided into three stages ([@b9-ppj-34-207]). The first stage is characterized by the invasion of the fungal cell wall into the intercellular space of a host cell. Hyphae with a branched network structure develop and exhibit intensive migration. The second stage is characterized by an increase in the population of hyphae in the cell wall and reduction in the number of intercellular spaces. Hyphae that continue to penetrate are also observed. The last stage is characterized by a decrease in the density of the branching network of hyphae, an increase in intercellular spaces, and a thickening of the host cell wall.
The transition from the first to the second stage and between the second and the third stage is regulated by microtubules ([@b18-ppj-34-207]). Hyphae of the primary hyphae of *P. viticola* were observed by [@b18-ppj-34-207]. Microtubules in a thin network structure aligned in the infection hyphae of *P. viticola* in the second stage. These data indicate that the hyphae growth of *P. viticola* is guided by microtubules. Microtubules are required for the induction of mitosis and the formation of cellular organelles and intracellular structures ([@b3-ppj-34-207]). The generation of microtubules by the chaperonin-assisted microtubule polymerization (CAMTP) is essential for hyphae growth. Subunit CAMTP interacts with tubulin during the assembly of the microtubules ([@b19-ppj-34-207]). Tubulin promotes the heterodimerization of chaperonin containing t-complex polypeptide 1 (COP1) and p23 with tubulin and the binding of COP1 and p23 to tubulin results in the conformational change of tubulin from a closed to an open state that converts the tubulin dimer into a protofilament ([@b12-ppj-34-207]). This conversion of tubulin from a closed to an open state causes the microtubule assembly. These microtubules are then condensed and develop a dense network, which initiates the second stage of infection. [@b15-ppj-34-207] reported that chitin in a thick cell wall is degraded in the second and the third stages of *P. viticola* infection. Thus, there is a direct correlation between the damage to chitin and the breaking of cell walls.
In this study, the effect of microtubules on the virulence of *P. viticola* was investigated. The virulence of *P. viticola* was assessed using virulence-related genes and the observation of the effect of hyphae on the host.
Material dan metode
Fungal strain, plant material, and maintenance
*P. viticola* race 2, obtained from the Korea *Plasmodiophora* Spore Collection, Korea Research Institute of Bioscience and Biotechnology (KRIBB), was grown on potato dextrose agar (PDA) for five days at 25°C. A *Vitis vinifera* (L.) 'Chardonnay' grafted onto *V. vinifera* (L.) 'Cabernet Sauvignon' cv 'Bungeong' rootstock was used as the host plant. The stock rootstock was purchased from Yonson Ltd. (Seoul, Korea) and maintained at 25°C in a greenhouse (6 h/24°C, 18 h/18°C).
Biological materials and infection tests
The effect of microtubules on the virulence of *P. viticola* was evaluated using the following plants and materials. Leaves from *V. vinifera* (L.) 'Chardonnay' were collected at four, six, and eight days post inoculation (dpi) with or without microtubules. The pathogen was prepared as previously described by [@b13-ppj-34-207]. For the microtubules preparation, hyphae were grown on PDA plates for seven days at 25°C and then transferred to a suspension containing 1.0 M colchicine for four hours. After a 1.0 M colchicine solution was removed by washing, the cultures were harvested in a tube and centrifuged at 12,000 rpm for 10 min at 4°C to obtain a hyphae fraction. The hyphae were purified by a three step procedure as previously described ([@b18-ppj-34-207]). The third step was repeated once. The virulence of the fungus was assessed by counting the number of lesions in the leaves at 12 dpi after inoculation. A leaf area with the lesions was selected to