Total protein extracts were immunoprecipitated (IP) with polyclonal antibody to AK6b. dicotyledonous plants. Among the T-DNA-encoded KIAA1516 proteins, 6b appears to play a role in the expression of herb genes related to cell proliferation and the modification of crown gall morphology, even though 6b protein itself is not required for the formation of crown galls (Garfinkel et al. 1981;Tinland et al. 1992). The6bgenes have amazing and unique effects on herb growth. They induce tumors on stems in a limited number of herb species, includingNicotiana (Z)-Capsaicin glauca(Hooykaas et al. 1988;Tinland et al. 1989;Kitakura et al. 2002), grapevine (Z)-Capsaicin (Huss et al. 1990), andNicotiana rustica(Garfinkel et al. 1981;Paulus et al. 1991); increase wound-induced division of cells inN. rustica(Tinland et al. 1992); promoteiaa- andipt-induced cell division (Hooykaas et al. 1988;Tinland et al. 1989); and enhance tumor formation or increase the size of crown galls induced onN. rusticathrough the action of theauxingenes and thecytokiningene (Tinland et al. 1989). Expression of6bgenes also elicits morphological and anatomical changes in tobacco plants, and modifies cotyledons, plants, petioles, stems, and roots (Tinland et al. 1990,1992;Helfer et al. 2003;Gremillon et al. 2004). Transgenic 6b tobacco plants develop enations, catacorollas, tubular leaves, fragmented leaf primordia, hyponastic petioles, and epinastic leaf veins (Clment et al. 2006). The predicted amino acid sequence of 6b is usually distantly related to the RolB family proteins, whose expression also results in aberrant growth and abnormal morphology of both roots and shoots (Spena et al. 1987;Levesque et al. 1988;Stieger et al. 2004). Protein 6b localizes to the nucleus and was reported to bind to several nuclear proteinssuch as NtSIP1 (Nicotiana tabacumSix-b-interacting protein 1) (Kitakura et al. 2002), NtSIP2 (Kitakura et al. 2008), and histone H3 (Terakura et al. 2007)and affects the expression of various herb genes in transgenicArabidopsis. TransgenicArabidopsisplants expressing theAK6bgene display leaf abnormality at early developmental stages, such as upward curling and serration of leaves, protrusion around the abaxial side of leaves, altered flower structure, and callus formation on hormone-free medium. Much effort has been expended to elucidate 6b functions at the cellular and molecular levels. So far, different hypotheses have been proposed to explain the effects of6bon the growth of herb cells. The6bgene could possess auxin-like and cytokinin-like activity by modulating auxin or cytokinin receptors, and promote activity of genes involved in auxin and cytokinin biosynthesis (Wabiko and Minemura 1996;Clment et al. 2006).6bmight induce plants to synthesize a phloem-mobile growth factor (enation factor) (Helfer et al. 2003). In addition, the6bgene has also (Z)-Capsaicin been proposed to modulate the metabolism of phenolic compounds (Glis et al. 2002,2004;Kakiuchi et al. 2006); induce changes in gene expression, resulting in phenylpropanoid changes (Kitakura et al. 2002;Terakura (Z)-Capsaicin et al. 2006); change osmotic controls required for normal and wound-induced cell growth (Clment et al. 2006); and cause the absorption, retention, and abnormal accumulation of sugars (Clment et al. 2007). Recent studies on6bfurther suggest that6bmight impact chromatin status as a chromatin chaperon or impact the expression of class IKNOX,CUC,IAA, and cell cycle-related genes as a transcriptional regulator of herb cell proliferation in tobacco andArabidopsis(Terakura et al. 2006,2007) Although much work has been done on 6b, the exact functional role of 6b at the molecular level remains unknown owing to the lack of structural motif information obtained from 6b sequences. To investigate the molecular mechanism of leaf abnormality elicited by 6b overexpression in plants, we report here thatArabidopsisplants overexpressing 6b display a microRNA (miRNA) deficiency phenotype. This phenotype can be attributed to the targeting of the miRNA pathway by targeting the miRNA processing machinery and (Z)-Capsaicin slicing machinery by 6b. In addition, we also statement high-resolution crystal structures of AK6b, AB6b, andArabidopsisADP ribosylation factor (ARF). Our data demonstrate that 6b adopts an ADP ribotransferase fold quite close to that of cholera toxin and displays ADP ribosylation activity in the presence ofArabidopsisARF and GTP. However, 6b has a completely different set of residues within the substrate-binding pocket for ADP ribosylation activity. == Results == == 6b interferes with miRNA pathways inArabidopsis == Many herb viruses are known to encode proteins that suppress RNA silencing and interfere with the miRNA pathway (Zhang et al. 2006b;Baumberger et al. 2007;Bortolamiol et al. 2007). Since T-DNA can be considered a DNA computer virus, we speculated that this 6b protein may also interfere with the miRNA pathway inArabidopsis. Careful comparison.
