Characterization of the Arabidopsis Lysine-Rich Arabinogalactan-Protein AtAGP17 Mutant (rat1) That Results in a Decreased Efficiency of Agrobacterium Transformation  

Abstract

Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. The Arabidopsis rat1 mutant, previously characterized as resistant to Agrobacterium tumefaciens root transformation, is due to a mutation in the gene for the Lys-rich AGP, AtAGP17. We show that the phenotype of rat1 correlates with down-regulation of AGP17 in the root as a result of a T-DNA insertion into the promoter of AGP17. Complementation of rat1 plants by a floral dip method with either the wild-type AGP17 gene or cDNA can restore the plant to a wild-type phenotype in several independent transformants. Based on changes in PR1 gene expression and a decrease in free salicylic acid levels upon Agrobacterium infection, we suggest mechanisms by which AGP17 allows Agrobacterium rapidly to reduce the systemic acquired resistance response during the infection process.Arabinogalactan-proteins (AGPs) are a family of complex proteoglycans widely distributed in plants. They are found in the extracellular matrix associated with the plasma membrane and cell wall (Knox, 1995;Du et al., 1996). Although the precise function(s) that AGPs perform is unknown, they have been implicated in diverse developmental roles, including differentiation, cell-cell recognition, and embryogenesis (Knox, 1996;Schultz et al., 1998;Majewski-Sawka and Nothnagel, 2000).Most studies investigating AGP expression and function have used an AGP-binding dye, b-D-glucosyl (b-D-Glc) Yariv reagent, and/or antibodies that recognize the carbohydrate epitopes of AGPs (for review, see Gaspar et al., 2001). These previous studies support a role for AGPs in plant cell growth and development; however, they do not inform us of the function of individual AGPs. The identification of AGP genes from Arabidopsis provides us with a wide range of tools to determine the function(s) of individual AGPs.To date, almost 50 genes encoding putative AGP protein backbones (hereafter referred to as AGP genes) have been identified in Arabidopsis (Schultz et al., 2002). These include the classical AGPs, those with Lys-rich domains, the arabinogalactan (AG)-peptides with short protein backbones, and the fasciclin-like AGPs ). Fasciclin-like AGPs are a class of chimeric AGPs that, in addition to AGP motifs, have fasciclin-like domains Johnson et al., 2003a). In addition, another approximately 50 glycosylphosphatidylinositol (GPI)-anchored proteins are likely to contain AG chains as part of larger proteins based on the presence of short Pro-, Ser-, Thr-, and Ala-rich regions containing noncontiguous Pro residues (Borner et al., 2002. Pro-, Ser-, Thr-, and Ala-rich regions contain noncontiguous Pro residues are referred to as AG-glycomodules because there is increasing evidence that these motifs direct the O-glycosylation of Hyp with type II AG chains (Goodrum et al., 2000;Zhao et al., 2002;Tan et al., 2003).Only a few AGP mutants have been identified to date. The haploinsufficient mutant, rat1 (resistant to Agrobacterium transformation), is resistant to transient and stable transforma...

The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium

“…Another potential explanation is that SA signaling also contributes in planta to resistance to Agrobacterium infection, but through an NPR1-independent pathway (10). It has been reported that Agrobacterium rapidly reduces the host systemic acquired resistance by downregulating PR gene expression and decreasing the free SA level by 40% within 1 h after infection (34). It is conceivable that this effect is mediated through NPR1; an inactivating mutation in NPR1 would abrogate the pathogen's ability to disable the plant defenses, resulting in enhanced host resistance.…”

“…However, at higher SA concentrations, inhibiting bacterial cell growth could be another strategy in the plant's defensive arsenal. Agrobacterium in turn appears to have evolved a mechanism to stymie the host defenses (34) and may even capitalize on the inhibitory effects of SA on vir gene expression to conserve Col-0 (WT) 33% (n ϭ 69) 33% (n ϭ 51) 24% (n ϭ 37) 3% (n ϭ 37) NahG 47% (n ϭ 30) 48% (n ϭ 54) 38% (n ϭ 29) 5% (n ϭ 38) npr1-1 17% (n ϭ 35) 2% (n ϭ 42) 9% (n ϭ 23) 0% (n ϭ 23) cpr5-2 22% (n ϭ 36) 18% (n ϭ 76) 3% (n ϭ 40) 0% (n ϭ 33) Col-0 (WT) 34% (n ϭ 61) 19% (n ϭ 63) LOX2 9% (n ϭ 171) 9% (n ϭ 155)…”

Agrobacterium-Mediated Genetic Transformation: History and Progress

“…AGPs were confirmed to be involved in A. tumefaciens transformation (Nam et al, 1999). Interestingly, AGP17 (rat1 mutant) appears to be involved in host defense reactions and signaling (Gaspar et al, 2004;Gelvin, 2010a). Other two rat mutans, ratT8 and ratT9, were identified to be mutated in the genes coding for receptor-like protein kinases (Zhu et al, 2003).…”

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

“…Lysine-Rich Arabinogalactan-Protein AtAGP17 [22] and an Arabidopsis glucomannan 4-betamannosyltransferase 9 gene AtCslA-09 [21] are both involved in the bacterial attachment to the plant cell wall. After attaching to the plant cell, the next important step of the infection is the transfer of the T-DNA into the host cell.…”

“…These studies identified approximately 40 plant genes (proteins) which are involved in Agrobacteriummediated transformation including, for example, the Arabinogalactan-Protein AtAGP17, the Reticulon domain proteins (BTI1-3), the VirE2 interacting proteins (Vip1 and Vip2), importins, histones and several other factors involved in the ubiquitin-proteasome complex [reviewed in 10,11]. Inactivation of such genes by insertion mutagenesis or gene silencing resulted in an attenuated tumor phenotype or even resistance to Agrobacterium transformation [7,8,[14][15][16][21][22][23], while their overexpression in transgenic plants increased their Agrobacterium sensitivity [15,24,25]. Although certain contributing genes, e. g. VIP1 and CSLA9, seem to be expressed constitutively [16,21], Agrobacteriuminfection induced the expression of a complete set of host genes involved in the transformation process [20].…”