Effect of the arbuscular mycorrhizal fungus Glomus intraradices on expression of cytoskeletal proteins in tomato roots S. Timonen and S.E. Smith Abstract: The expression patterns of the cytoskeletal proteins α-, β-, and γ-tubulin, actin, and myosin were investigated in young tomato roots and older roots at different intensities of mycorrhizal colonization. The relative level of cytoskeletal proteins was estimated by protein blotting and immunostaining. The contribution of plant α-, β-, and γ- tubulin to the total protein pool was higher in uncolonized 2-week-old roots than in 10-week-old roots, whereas the contribution of actin remained constant. The level of plant tubulin expression was clearly higher in mycorrhizal root systems than in uncolonized older root systems. These results indicate that tubulins are more involved in plant cell dif- ferentiation than actin. Myosin of approximately 230 kDa was expressed in the roots of 10-week-old wild-type tomato but not in young or mycorrhizal tomatoes. In contrast, a smaller ca. 170 kDa myosin was consistently present in all root samples. Indirect immunofluorescence microscopy showed that plant myosin was located particularly along the periarbuscular membranes surrounding the arbuscule branches. In uncolonized roots, myosin was associated mainly with membranes adjacent to plant cell walls. These data provide novel evidence that myosin expression and localization in root cells responds to mycorrhizal colonization. Key words: actin, myosin, arbuscular mycorrhizal fungi, protein expression, tubulin. Résumé : Les auteurs ont étudié les patrons d’expression des protéines du cytosquelette; tubulines α, β et γ actine et myosine, chez des racines de tomate, jeunes et plus âgées, avec différents degrés de colonisation mycorhizienne. Ils ont évalué la teneur relative en protéines du cytosquelette par transfert et immuno-coloration. La contribution des tubulines la plante des tubulines α, β et γ au pool total des protéines est plus élevée chez les racines de tomate non-colonisées âgées de 2 semaines, que chez les racines âgées de 10 semaines, alors que la contribution de l’actine demeure cons- tante. Le degré d’expression de la tubuline de la plante est nettement plus élevé dans les systèmes mycorhiziens que chez les systèmes racinaires âgés non colonisés. Ces résultats indiquent qu’une plus grande implication des tubulines et de l’actine dans la différenciation cellulaire. Une myosine d’environ 230 kDa s’exprime dans les racines de tomate âgées du type sauvage de 10 semaines, mais pas dans les racines jeunes ou les racines mycorhizées de tomate. Au con- traire, on retrouve de façon constante une myosine plus petite, ca. 170 kDa, dans toutes les racines examinées. La mi- croscopie en fluorescence indirecte montre que la myosine de la plante est localisée surtout le long de la membrane péri-arbusculaire entourant les branches des arbuscules. Chez les racines non-colonisées, la myosine est associée surtout aux membranes adjacentes aux parois cellulaires de la plante. Ces données présentent une nouvelle preuve que l’expression et la localisation de la myosine dans les cellules racinaires réagissent à la colonisation mycorhizienne. Mots clés : actine, myosine, champignons arbusculaires mycorhiziens, expression de la protéine, tubuline. [Traduit par la Rédaction] Timonen and Smith 182 Introduction The cytoskeleton is a protein-based organisation and transportation system present in all eukaryotic cells. The most studied cytoskeletal proteins are the filament-forming actin and tubulin. Together with their associated proteins, these cytoskeletal filaments control most cellular move- ments, nuclear and cellular division, as well as placement of cell contents. The profound cellular changes that occur dur- ing arbuscular mycorrhizal (AM) colonization of the roots could not take place without involvement of the cytoskeleton of both partners (see Timonen and Peterson (2002) and the references therein). Indeed significant reor- ganisation of both actin filaments and microtubules of the plant partner occurs during AM colonization (Genre and Bonfante 1997, 1998). γ-Tubulin, a protein that triggers α- and β-tubulin assembly into microtubules, has been shown to closely associate with the plant membrane surrounding the terminal branches of arbuscules (Genre and Bonfante 1999). Vesicle-coating protein, clathrin, associating with micro- tubules has also been shown to congregate along plant mem- 176 Can. J. Bot. 83: 176–182 (2005) doi: 10.1139/B04-160 © 2005 NRC Canada Received 16 July 2004. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 8 February 2005. S. Timonen 1,2 and S.E. Smith. Centre for Soil-Plant Interactions, Soil and Land Systems, School of Earth and Environmental Sciences, Waite Campus, The University of Adelaide, Adelaide, South Australia 5005, Australia. 1 Corresponding author (e-mail: sari.timonen@helsinki.fi). 2 Present address: Department of Applied Biology, P.O. Box 27 (Latokartanonkaari 7), 00014 University of Helsinki, Helsinki, Finland.