![]() (2008) subsequently detected such interactions in yeast, in planta, and in vitro. Early work indicated that VirE2 does not interact with Arabidopsis importin alpha-1 (IMPa-1, also known as AtKAPα) in yeast ( Ballas and Citovsky, 1997), although Bhattacharjee et al. VirE2 possesses a weak putative nuclear localization signal (NLS) sequence which does not bind strongly to importin α protein ( Citovsky et al., 1994 Chang et al., 2014). However, other studies showed exclusively cytoplasmic localization of VirE2 ( Bhattacharjee et al., 2008 Grange et al., 2008 Lee et al., 2008 Sakalis et al., 2013 Shi et al., 2014). Previous studies indicated that β-glucoronidase-(GUS) or YFP-tagged VirE2 localizes to the plant nucleus ( Citovsky et al., 1992, 1994 Tzfira and Citovsky, 2001). thaliana VIP1 overexpressing lines showed no effect on transformation susceptibility ( Shi et al., 2014), suggesting that VIP1 is not important for Agrobacterium-mediated transformation. However, quantitative transformation assays with the vip1-1 mutant and with 59 A. Overexpression of VIP1 in tobacco resulted in increased transformation susceptibility, suggesting that VIP1 plays a role in transformation ( Tzfira et al., 2001). Previous studies using transgenic tobacco lines expressing antisense constructs targeting VIP1, and the Arabidopsis thaliana T-DNA insertion mutant vip1-1, found that these plants showed decreased stable transformation compared to that of wild-type plants ( Tzfira et al., 2001 Li et al., 2005). The importance and role of VIP1 in Agrobacterium-mediated transformation are controversial ( Tzfira et al., 2001 Shi et al., 2014). VIP1 may also be involved in sulfur utilization, starch accumulation, osmosensory signaling, and touch-induced root waving ( Ishida et al., 2004 Wu et al., 2010 Tsugama et al., 2012, 2014, 2016 Chen et al., 2015). VIP1 may subsequently bind to VIP1 response elements (VREs) to activate transcription of its target genes ( Pitzschke et al., 2009). Phosphorylation of VIP1 on serine 79 by MPK3 results in the import of VIP1 into the plant nucleus ( Djamei et al., 2007). VIP1, a bZIP transcription factor which is a target of the mitogen-activated protein kinase 3 (MPK3), is thought to be involved in plant defense responses ( Djamei et al., 2007 Pitzschke et al., 2009). In addition to this structural role, VirE2 interacts with a number of plant proteins including VirE2-interacting protein 1 (VIP1 Tzfira et al., 2001) and VIP2 ( Anand et al., 2007). The effector protein VirE2 has non-specific single-stranded DNA-binding activity and is thought to coat single-stranded T-DNA (T-strands) after entry into the plant cell ( Citovsky et al., 1992), protecting T-strands from nucleolytic degradation ( Yusibov et al., 1994 Rossi et al., 1996). Agrobacterium-mediated plant genetic transformation involves mobilization of transferred-DNA (T-DNA) and five virulence proteins (VirD2, VirE2, VirE3, VirD5, and VirF) from the bacterium into a plant cell ( Gelvin, 2003, 2012). Virulent strains of the soil bacterium Agrobacterium tumefaciens cause the tumorigenic disease crown gall. vip1 mutant and VIP1-SRDX plants show increased tolerance to growth in salt, indicating a role for VIP1 in response to salt stress. The germination of vip1 mutant and VIP1-SRDX seeds is sensitive to exogenous ABA, suggesting a role for VIP1 in response to ABA. cinerea susceptibility is dependent on abscisic acid (ABA) which is also important for abiotic stress responses. syringae infection, suggesting a role for VIP1 in B. vip1 mutant and VIP1-SRDX plants show increased susceptibility to B. Because VIP1 is involved in innate immune response signaling, we tested the susceptibility of vip1 mutant and VIP1-SRDX plants to Pseudomonas syringae and Botrytis cinerea. All VIP1-SRDX transgenic lines showed wild-type levels of transformation, indicating that neither VIP1 nor its homologs are required for Agrobacterium-mediated transformation. Because of potential functional redundancy among VIP1 homologs, we tested transgenic lines expressing VIP1 fused to a SRDX repression domain. The transformation susceptibility of vip1-2 and wild-type plants is similar. We generated the CRISPR/Cas mutant vip1-2 that lacks this domain. Previously tested vip1-1 mutant plants produce a truncated protein containing the crucial bZIP DNA-binding domain. The bZIP transcription factor VIP1 interacts with the Agrobacterium virulence protein VirE2, but the role of VIP1 in Agrobacterium-mediated transformation remains controversial. 3Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN, United States.2Laboratory of Crop Physiology, Research Faculty of Agriculture, Hokkaido University, Sapporo, Japan.1Department of Biological Sciences, Purdue University, West Lafayette, IN, United States.Rachelle Lapham 1, Lan-Ying Lee 1, Daisuke Tsugama 2, Sanghun Lee 3, Tesfaye Mengiste 3 and Stanton B. ![]()
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